Solid support assay systems and methods utilizing non-standard bases

ABSTRACT

Solid support assays using non-standard bases are described. A capture oligonucleotide comprising a molecular recognition sequence is attached to a solid support and hybridized with a target. In some instances, the molecular recognition sequence includes one or more non-standard bases and hybridizes to a complementary tagging sequence of the target oligonucleotide. In other instances, incorporation of a non-standard base (e.g., via PCR or ligation) is used in the assay.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.11/490,319 filed Jul. 20, 2006, now U.S. Pat. No. 8,217,160, which is acontinuation of U.S. application Ser. No. 11/284,307, filed on Nov. 19,2005, now U.S Pat. No. 7,892,796; which is a continuation of U.S.application Ser. No. 09/977,615, filed on Oct. 15, 2001, now U.S. Pat.No. 6,977,161; which claims the benefit of U.S. Provisional ApplicationSer. Nos. 60/293,259, filed on May 22, 2001; 60/282,831, filed on Apr.10, 2001; and 60/240,397, filed on Oct. 14, 2000. U.S. application Ser.No. 09/977,615 is a continuation-in-part of U.S. application Ser. No.09/861,292, filed on May 18, 2001, now U.S. Pat. No. 7,422,850, whichclaims the benefit of U.S. Provisional Application Ser. Nos. 60/282,831,filed on Apr. 10, 2001; 60/240,398, filed on Oct. 14, 2000; and60/205,712, filed on May 19, 2000. The aforementioned applications areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

A variety of different methods have been developed to assayoligonucleotides, including DNA or RNA fragments. Such assays aretypically directed to determining whether a sample includesoligonucleotides having a particular target oligonucleotide sequence. Insome instances, oligonucleotide sequences differ by only a fewnucleotides, as in the case of many allelic sequences. Single nucleotidepolymorphisms (SNPs) refer to alleles that differ by a singlenucleotide. Even this single nucleotide difference can, at least in someinstances, change the associated genetic response or traits.Accordingly, to determine which allele is present in a sample, the assaytechnique must be sufficiently sensitive to distinguish between closelyrelated sequences.

Many assay techniques include multiple components, each of whichhybridizes to other component(s) in the assay. Non-specifichybridization between components (i.e., the hybridization of twonon-complementary sequences) produces background noise in the assay. Forexample, closely related, but not identical, sequences can formimperfect duplexes in which base pairing is interrupted at positionswhere the two single strands are not complementary. Non-specifichybridization increases when the hybridizing components have similarsequences, as would be the case, for example, for many alleles andparticularly for SNP alleles. Thus, for example, hybridization assays todetermine which allele is present in a sample would benefit from methodsthat reduce non-specific hybridization or reduce the impact ofnon-specific hybridization on the assay.

BRIEF SUMMARY OF THE INVENTION

Generally, the present invention relates to methods, kits, andcompositions for assaying oligonucleotides. In addition, the inventionrelates to methods, kits, and compositions for assaying oligonucleotidesusing non-standard bases. One embodiment provides a method of assayingan analyte-specific sequence. A capture oligonucleotide comprising amolecular recognition sequence having at least one non-standard basecoupled to a support (e.g., a single solid support, such as a chip orwafer, or a particulate support) is contacted with a sample undersuitable hybridizing conditions to hybridize to a targetoligonucleotide, if present in the sample. The target oligonucleotidecomprises a tagging sequence complementary to the molecular recognitionsequence of the capture oligonucleotide and the analyte-specificsequence or a complement of the analyte-specific sequence. Hybridizationof the target oligonucleotide to the capture oligonucleotide isdetected.

Another embodiment provides another method of assaying ananalyte-specific sequence. A capture oligonucleotide coupled to asupport and comprising a molecular recognition sequence that is the sameas or complementary to at least a portion of the analyte-specificsequence is contacted with a sample under hybridizing conditions tohybridize to a target oligonucleotide. The target oligonucleotidecomprises a tagging sequence comprising at least one non-standard baseand the analyte-specific sequence or a complement of theanalyte-specific sequence. The capture oligonucleotide is enzymaticallyextended using the target oligonucleotide as a template and acomplementary non-standard base is incorporated opposite thenon-standard base of the tagging sequence. A reporter group is alsoincorporated into an extended portion of the capture oligonucleotide.Hybridization of the target oligonucleotide to the captureoligonucleotide is detected.

Yet another embodiment provides another method of assaying ananalyte-specific sequence. An analyte having the analyte-specificsequence is contacted with a first primer and a second primer. The firstprimer comprises a tagging sequence and a sequence complementary to afirst sequence of the analyte. The second primer comprises a sequencecomplementary to a second sequence of the analyte and a non-standardbase. The first and second primers are enzymatically extended to form atarget oligonucleotide and a second oligonucleotide, respectively. Oneof the target oligonucleotide and the second oligonucleotide comprisesthe analyte-specific sequence, and the other comprises a sequencecomplementary to the analyte-specific sequence. Extension of the firstprimer is substantially halted when the non-standard base of the secondprimer is encountered. A non-standard base complementary to thenon-standard base of the second primer is incorporated into the extendedfirst primer opposite the non-standard base of the second primer. Acapture oligonucleotide molecular recognition sequence that is the sameas or complementary to at least a portion of the analyte-specificsequence coupled to a support is contacted with the targetoligonucleotide under hybridizing conditions to hybridize to the targetoligonucleotide comprising a tagging sequence and the analyte-specificsequence or complement thereof. Hybridization of the targetoligonucleotide to the capture oligonucleotide is detected.

Other embodiments include kits for applying the methods described above.The kits include support(s) and capture oligonucleotides. The kits alsoinclude the target oligonucleotides or components for making the targetoligonucleotides from an analyte. Such components can include, forexample, a polymerase and first and second primers that arecomplementary to sequences of the analyte, where either the first orsecond primers include the tagging sequence. For some methods, the kitcan also include a non-standard base or nucleotide triphosphate of anon-standard base for incorporation.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The Figures and the detailed description which follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1 displays chemical structures for a number of non-standard bases,where A is the point of attachment to a polymeric backbone, X is N orC—Z, Y is N or C—H, and Z is H or a substituted or unsubstituted alkylgroup;

FIGS. 2A and 2B schematically illustrate two examples of oligonucleotidehybridization to a solid support, according to the invention;

FIG. 3 illustrates steps in a first assay for an analyte-specificsequence, according to the invention;

FIG. 4 illustrates steps in a second assay for an analyte-specificsequence, according to the invention;

FIG. 5 illustrates steps in a third assay for an analyte-specificsequence, according to the invention;

FIG. 6 illustrates steps in a fourth assay for an analyte-specificsequence, according to the invention;

FIG. 7 illustrates steps in a fifth assay for an analyte-specificsequence, according to the invention;

FIG. 8 illustrates steps in a sixth assay for an analyte-specificsequence, according to the invention;

FIG. 9 illustrates steps in a seventh assay for an analyte-specificsequence, according to the invention;

FIG. 10 illustrates steps in an eighth assay for an analyte-specificsequence, according to the invention;

FIG. 11 illustrates steps in a ninth assay for an analyte-specificsequence, according to the invention;

FIG. 12 is a 3D surface map illustrating, for 98 molecular recognitionsequences (y-axis), the hybridization of complementary tagging sequences(x-axis) for each of the 100 molecular recognition sequences;

FIG. 13 is a 3D surface map illustrating, for 50 molecular recognitionsequences (y-axis), the hybridization of complementary tagging sequences(x-axis) for each of the 50 molecular recognition sequences;

FIG. 14 is a graph illustrating results from an assay of alleles,according to the invention;

FIGS. 15A and 15B are graphs illustrating results from another assay ofalleles, according to the invention; and

FIG. 16 illustrates steps in a tenth assay for an analyte-specificsequence, according to the invention.

FIG. 17 is a graph of results from an assay of alleles, according to theinvention.

Although the invention is amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in detail. It should beunderstood, however, that the invention is not limited to the particularembodiments described. On the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to assays and methods of assayingoligonucleotides. In particular, the present invention is directed toassays and methods of assaying oligonucleotides using one or morenon-standard bases. Although the present invention is not so limited, anappreciation of various aspects of the inventions described herein willbe gained through the discussion provided below. Other related assaymethods for use with non-standard bases are described in U.S. PatentProvisional Application Ser. No. 60/240,397, filed Oct. 14, 2000.

As used herein, “nucleic acids” include polymeric molecules such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleicacid (PNA), or any sequence of what are commonly referred to as basesjoined by a chemical backbone where the bases have the ability to formbase pairs or hybridize with a complementary chemical structure.Suitable non-nucleotidic chemical backbones include, for example,polyamide and polymorpholino backbones. The term “nucleic acids”includes oligonucleotide, nucleotide, or polynucleotide sequences, andfragments or portions thereof. The nucleic acid can be provided in anysuitable form, e.g., isolated from natural sources, recombinantlyproduced, or artificially synthesized, can be single- ordouble-stranded, and can represent the sense or antisense strand.

The term “oligonucleotide” refers generally to short chain (e.g., lessthan about 100 nucleotides in length, and typically 6 to 50 nucleotidesin length) nucleic acid sequences as prepared using techniques presentlyavailable in the art such as, for example, solid support nucleic acidsynthesis, DNA replication, reverse transcription, restriction digest,run-off transcription, or the like. The exact size of theoligonucleotide will typically depend upon a variety of factors, whichin turn will depend upon the ultimate function or use of theoligonucleotide.

A “sequence” refers to an ordered arrangement of nucleotides.

The term “sample” includes a specimen or culture (e.g., microbiologicalcultures), as well as biological samples, samples derived frombiological fluids, and samples from non-biological sources.

The term “analyte” refers to a nucleic acid suspected to be in a sample.The analyte is the object of the assay (e.g., the assay determines thepresence, absence, concentration, or amount of the analyte in thesample). The analyte can be directly or indirectly assayed. In at leastsome embodiments involving indirect assay, the analyte, if present inthe sample, is used as a template to form target oligonucleotides using,for example, PCR techniques. The target oligonucleotides are thenassayed to indicate the presence, absence, concentration, or amount ofthe analyte in the sample.

The term “target oligonucleotide” refers to oligonucleotides that areactually assayed during an assay procedure. The target oligonucleotidecan be, for example, an analyte or it can be an oligonucleotidecontaining an analyte-specific sequence that is the same as orcomplementary to a sequence of the analyte. For example, the targetoligonucleotide can be a product of PCR amplification of an analyte or aportion of an analyte.

The term “capture oligonucleotide” refers to an oligonucleotide having amolecular recognition sequence and coupled to a solid surface tohybridize with a target oligonucleotide having a tagging sequence or ananalyte specific sequence complementary to the molecular recognitionsequence, thereby capturing the target oligonucleotide on the solidsurface.

A “molecular recognition sequence” as used herein is an oligonucleotidesequence complementary to the tagging sequence or to theanalyte-specific sequence of a target oligonucleotide.

As used herein, the terms “complementary” or “complementarity,” whenused in reference to nucleic acids (i.e., a sequence of nucleotides suchas an oligonucleotide or a target nucleic acid), refer to sequences thatare related by base-pairing rules. For natural bases, the base pairingrules are those developed by Watson and Crick. For non-standard bases,as described herein, the base-pairing rules refer to the formation ofhydrogen bonds in a manner similar to the Watson-Crick base pairingrules or the formation of specific base pairs by hydrophobic, entropic,or van der Waals forces. As an example, for the sequence “T-G-A”, thecomplementary sequence is “A-C-T.” Complementarity can be “partial,” inwhich only some of the bases of the nucleic acids are matched accordingto the base pairing rules. Alternatively, there can be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between the nucleic acid strands affects the efficiencyand strength of hybridization between the nucleic acid strands.

The term “hybridization” is used in reference to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is influenced by such factors as the degree of complementaritybetween the nucleic acids, stringency of the hybridization conditionsinvolved, the melting temperature (T_(m)) of the formed hybrid, and theG:C ratio within the nucleic acids.

Assays are performed to determine whether a sample includes an analytehaving a particular nucleic acid sequence (or its complement). Thisnucleic acid sequence will be referred to as the “analyte-specificsequence”. In at least some instances, the original sample is notdirectly assayed. Instead, the analyte, if present, is cloned oramplified (e.g., by PCR techniques) to provide an assay sample with adetectable amount of a target oligonucleotide that contains theanalyte-specific sequence. Other techniques for amplification include,for example, nucleic acid sequence based amplification (NASBA, e.g.,Guatelli, et al., Proc. Nat'l. Acad. Sci. 87, 1874 (1990), incorporatedherein by reference), strand displacement amplification (SDA, e.g.,Walker, et al., Proc. Nat'l. Acad. Sci. 89, 392-96 (1992), incorporatedherein by reference), ligase chain reaction (LCR, e.g., Kalin, et al.,Mutat. Res., 283, 119-23 (1992), incorporated herein by reference),transcription mediated amplification (TMA, e.g., La Rocco, et al., Eur.J. Clin. Microbiol. Infect. Dis., 13, 726-31 (1994), incorporated hereinby reference), and rolling circle amplification (RCA, e.g., Lizardi, etal., Nat. Genet., 19, 225-32 (1998), incorporated herein by reference).At least a portion of the target oligonucleotide typically correspondsto either a) the analyte, b) a portion of the analyte, c) a complementof the analyte, or d) a complement of a portion of the analyte.Detection of the target oligonucleotide by the assay indicates presenceof the analyte in the original sample.

In general, an assay system for detecting one or more analyte-specificsequences includes a solid support (e.g., a chip, wafer, or a collectionof solid particles). Capture oligonucleotides are disposed on the solidsupport in a manner which permits identification of the captureoligonucleotide (e.g., by position on a chip or wafer or by uniquecharacteristic of particles to which particular capture oligonucleotidesare attached). The capture oligonucleotides include a molecularrecognition sequence. Different capture oligonucleotides with differentmolecular recognition sequences are used to detect differentanalyte-specific sequences. Using these different captureoligonucleotides, a single assay system can be designed to analyze asample for multiple analyte-specific sequences.

Target oligonucleotides containing the analyte-specific sequences arebrought into contact with the capture oligonucleotides. In addition tothe analyte-specific sequence, the target oligonucleotides also eachinclude a tagging sequence. A particular tagging sequence is associatedwith each analyte-specific sequence. The tagging sequence is generallycomplementary to one of the molecular recognition sequences. Thus, underhybridization conditions, the target oligonucleotides hybridize with theappropriate capture oligonucleotides. Alternatively, in certain methodsof the present invention, the analyte-specific sequence may becomplementary to one of the molecular recognition sequences.

The target oligonucleotide or its complement typically includes areporter or a coupling agent for attachment of a reporter. Observationof the solid support to determine the presence or absence of thereporter associated with a particular capture oligonucleotide indicateswhether a particular analyte-specific sequence is present in the sample.Suitable reporters include, without limitation, biotin, fluorescents,chemilluminescents, digoxigenin, spin labels, radio labels, DNA cleavagemoieties, chromaphors or fluoraphors. Examples of suitable couplingmoieties include, but are not limited to, amines, thiols, hydrosines,alcohols or alkyl groups.

Examples of suitable assay systems are schematically illustrated inFIGS. 2A and 2B. In these assays, capture oligonucleotides 100 a, 100 bare coupled to a solid support 120, such as, for example, a single solidsubstrate 120 a (e.g., a chip or wafer) or one of a number of solidparticles 120 b. Typically, at least one of the capture oligonucleotides(e.g., capture oligonucleotide 100 a) has a molecular recognitionsequence 102 that is complementary to a tagging sequence 112 of a targetoligonucleotide 110 so that, under hybridization conditions, the targetoligonucleotide 110 hybridizes to the capture oligonucleotide 100 a.

Although assays can be prepared with all of the capture oligonucleotideshaving the same global molecular recognition sequence, typically, two ormore different groups of capture oligonucleotides 100 a, 100 b are used.Each group of capture oligonucleotides has a different molecularrecognition sequence. On a single solid substrate, each group of captureoligonucleotides are typically disposed on a particular region orregions of the substrate such that the region(s) is/are associated witha particular molecular recognition sequence. When a particle support isused, each group of capture oligonucleotides 100 a, 100 b is disposed onat least one group of particles 120 b, 120 c having a uniquecharacteristic such that the capture oligonucleotide of a particularparticle is determined from the characteristic of the particle to whichit is attached. Such assays can be used to, for example, a) determinewhich allele is present in a sample by associating different captureoligonucleotides (and different regions of a substrate or differentgroups of particles) with each allele, b) assay for multiple related orunrelated oligonucleotides or c) both. As illustrated in FIGS. 2A and2B, the target oligonucleotide preferentially hybridizes to acorresponding capture oligonucleotide permitting determination of thepresence or absence of an analyte-specific sequence by observation ofthe presence or absence of a target oligonucleotide on a particularspatial position of the single support (FIG. 2A) or attached to aparticular group of particles (FIG. 2B).

An additional component of the assay system is a reporter 130 thatcouples to the target oligonucleotide 110 (or its complement 120), asdescribed below. The reporter 130 is the component of the assay that issubsequently detected by a detection technique (e.g., by colorimetric,fluorescence, electrophoretic, electrochemical, spectroscopic,chromatographic, densitometric, or radiographic techniques) to indicatethe presence or concentration of the target oligonucleotide. Thereporter will typically be determined by the detection technique (e.g.,fluorophore reporters for fluorescent techniques and radio-labels forradiographic techniques.)

In some assays, one or both of the capture oligonucleotide and thetarget oligonucleotide include at least one non-standard base. The useof non-standard base(s) can improve the specificity of an assay thatincludes hybridization because non-standard bases preferentiallyhybridize to other complementary non-standard bases. The use of longeroligonucleotides can also increase the rate of specific hybridization.The hybridization of nucleic acids generally includes the sampling ofabout three to four bases for complete complementarity. These formnucleation sites. If a nucleation site is found, the hybridizationproceeds down the strand. If the bases down the strand are notcomplementary, then the two strands release. Because the nucleationprocess takes time, the possibilities of finding a nucleation site whennon-standard bases are used is reduced, thereby reducing the number ofsampling steps needed to find a complete complement.

Alternatively, the non-standard bases are used to direct the addition ofanother non-standard base into a sequence (using, for example, PCRtechniques). The added non-standard base can include a reporter or acoupling agent to which a reporter can be attached, thereby, permittingthe highly selective incorporation of a reporter group for detection ofthe target oligonucleotide.

Oligonucleotides and Bases

DNA and RNA are oligonucleotides that include deoxyriboses or riboses,respectively, coupled by phosphodiester bonds. Each deoxyribose orribose includes a base coupled to a sugar. The bases incorporated innaturally-occurring DNA and RNA are adenosine (A), guanosine (G),thymidine (T), cytidine (C), and uridine (U). These five bases are“natural bases”. According to the rules of base pairing elaborated byWatson and Crick, the natural bases can hybridize to formpurine-pyrimidine base pairs, where G pairs with C and A pairs with T orU. These pairing rules facilitate specific hybridization of anoligonucleotide with a complementary oligonucleotide.

The formation of these base pairs by the natural bases is facilitated bythe generation of two or three hydrogen bonds between the two bases ofeach base pair. Each of the bases includes two or three hydrogen bonddonor(s) and hydrogen bond acceptor(s). The hydrogen bonds of the basepair are each formed by the interaction of at least one hydrogen bonddonor on one base with a hydrogen bond acceptor on the other base.Hydrogen bond donors include, for example, heteroatoms (e.g., oxygen ornitrogen) that have at least one attached hydrogen. Hydrogen bondacceptors include, for example, heteroatoms (e.g., oxygen or nitrogen)that have a lone pair of electrons.

The natural bases, A, G, C, T, and U, can be derivatized by substitutionat non-hydrogen bonding sites to form modified natural bases. Forexample, a natural base can be derivatized for attachment to a supportby coupling a reactive functional group (e.g., thiol, hydrazine,alcohol, or amine) to a non-hydrogen bonding atom of the base. Otherpossible substituents include biotin, digoxigenin, fluorescent groups,and alkyl groups (e.g., methyl or ethyl).

Non-standard bases, which form hydrogen-bonding base pairs, can also beconstructed as described, for example, in U.S. Pat. Nos. 5,432,272,5,965,364, 6,001,983, and 6,037,120 and U.S. patent application Ser. No.08/775,401, all of which are incorporated herein by reference. By“non-standard base” it is meant a base other than A, G, C, T, or U thatis susceptible of incorporation into an oligonucleotide and which iscapable of base-pairing by hydrogen bonding, or by hydrophobic,entropic, or van der Waals interactions to form base pairs with acomplementary base. FIG. 1 illustrates several examples of suitablebases and their corresponding base pairs. Specific examples of thesebases include the following bases in base pair combinations(iso-C/iso-G, K/X, H/J, and M/N):

where A is the point of attachment to the sugar or other portion of thepolymeric backbone and R is H or a substituted or unsubstituted alkylgroup. It will be recognized that other non-standard bases utilizinghydrogen bonding can be prepared, as well as modifications of theabove-identified non-standard bases by incorporation of functionalgroups at the non-hydrogen bonding atoms of the bases. To designatethese non-standard bases in FIGS. 3 to 9, the following symbols will beused: X indicates iso-C and Y indicates iso-G.

The hydrogen bonding of these non-standard base pairs is similar tothose of the natural bases where two or three hydrogen bonds are formedbetween hydrogen bond acceptors and hydrogen bond donors of the pairingnon-standard bases. One of the differences between the natural bases andthese non-standard bases is the number and position of hydrogen bondacceptors and hydrogen bond donors. For example, cytosine can beconsidered a donor/acceptor/acceptor base with guanine being thecomplementary acceptor/donor/donor base. Iso-C is anacceptor/acceptor/donor base and iso-G is the complementarydonor/donor/acceptor base, as illustrated in U.S. Pat. No. 6,037,120,incorporated herein by reference.

Other non-standard bases for use in oligonucleotides include, forexample, naphthalene, phenanthrene, and pyrene derivatives as discussed,for example, in Ren et al., J. Am. Chem. Soc. 118, 1671 (1996) andMcMinn et al., J. Am. Chem. Soc. 121, 11585 (1999), both of which areincorporated herein by reference. These bases do not utilize hydrogenbonding for stabilization, but instead rely on hydrophobic, entropic, orvan der Waals interactions to form base pairs.

Solid Supports

The assay is carried out, at least in part, using a solid support.Generally, the capture oligonucleotides are coupled to or otherwisedisposed on a surface of the support. A variety of different supportscan be used. In some embodiments, the solid support is a single solidsupport, such as a chip or wafer, or the interior or exterior surface ofa tube, cone, or other article. The solid support is fabricated from anysuitable material to provide an optimal combination of such desiredproperties as stability, dimensions, shape, and surface smoothness.Preferred materials do not interfere with nucleic acid hybridization andare not subject to high amounts of non-specific binding of nucleicacids. Suitable materials include biological or nonbiological, organicor inorganic materials. For example, the master array can be fabricatedfrom any suitable plastic or polymer, silicon, glass, ceramic, or metal,and can be provided in the form of a solid, resin, gel, rigid film, orflexible membrane. Suitable polymers include, for example, polystyrene,poly(alkyl)methacrylate, poly(vinylbenzophenone), polycarbonate,polyethylene, polypropylene, polyamide, polyvinylidenefluoride, and thelike. Preferred materials include polystyrene, glass, and silicon.

In some embodiments, the single solid support 300 is divided intoindividual regions 310 with capture oligonucleotides disposed on thesupport in each region, as illustrated in FIG. 3. In each of the regionsor on each particle support, the capture oligonucleotides havepredominantly (e.g., at least 75%) the same molecular recognitionsequence. Preferably, substantially all (e.g., at least 90% and, morepreferably, at least 99%) of the capture oligonucleotides have the samemolecular recognition sequence in each region or on each particlesupport. The capture oligonucleotides of different regions typicallyhave different sequences, although in some instances, the same captureoligonucleotides can be used in two or more regions, for example, as acontrol or verification of results.

A solid support with different regions can form a regular or irregulararray for testing samples and determining the presence or absence of anumber of different analyte-specific sequences. For example, an arraycan be formed to test for 10, 100, 1000 or more differentanalyte-specific sequences.

Dimensions of the solid support are determined based upon such factorsas the desired number of regions and the number of analyte-specificsequences to be assayed. As an example, a solid support can be providedwith planar dimensions of about 0.5 cm to about 7.5 cm in length, andabout 0.5 cm to about 7.5 cm in width. Solid supports can also be singlyor multiply positioned on other supports, such as microscope slides(e.g., having dimensions of about 7.5 cm by about 2.5 cm). Thedimensions of the solid support can be readily adapted for a particularapplication.

Other types of solid supports can be used. In some embodiments, thesolid support is a particulate support. In these embodiments, thecapture oligonucleotides are coupled to particles. Typically, theparticles form groups in which particles within each group have aparticular characteristic, such as, for example, color, fluorescencefrequency, density, size, or shape, which can be used to distinguish orseparate those particles from particles of other groups. Preferably, theparticles can be separated using techniques, such as, for example, flowcytometry.

As contemplated in the invention, the particles can be fabricated fromvirtually any insoluble or solid material. For example, the particlescan be fabricated from silica gel, glass, nylon, resins, Sephadex™,Sepharose™, cellulose, magnetic material, a metal (e.g., steel, gold,silver, aluminum, copper, or an alloy) or metal-coated material, aplastic material (e.g., polyethylene, polypropylene, polyamide,polyester, polyvinylidenefluoride (PVDF)) and the like, and combinationsthereof. Examples of suitable micro-beads are described, for example, inU.S. Pat. Nos. 5,736,330, 6,046,807, and 6,057,107, all of which areincorporated herein by reference. Examples of suitable particles areavailable, for example, from Luminex Corp., Austin, Tex.

In one embodiment, the particulate supports with associated captureoligonucleotides are disposed in a holder, such as, for example, a vial,tube, or well. The target oligonucleotide is added to the holder and theassay is conducted under hybridization conditions. The particulatesupports are then separated on the basis of the unique characteristicsof each group of supports. The groups of supports are then investigatedto determine which support(s) have attached target oligonucleotides.Optionally, the supports can be washed to reduce the effects ofcross-hybridization. One or more washes can be performed at the same ordifferent levels of stringency, as described below. As another optionalalternative, prior to contact with the support(s) and captureoligonucleotides, the solution containing target oligonucleotides can besubjected to, for example, size exclusion chromatography, differentialprecipitation, spin columns, or filter columns to remove primers thathave not been amplified or to remove other materials that are not thesame size as the target oligonucleotides.

In some embodiments, multiple holders (e.g., vials, tubes, and the like)are used to assay multiple samples or have different combinations ofcapture oligonucleotides (and associated supports) within each holder.As another alternative, each holder can include a single type of captureoligonucleotide (and associated support).

As another example, the support can be a group of individual supportsurfaces that are optionally coupled together. For example, the supportcan include individual optical fibers or other support members that areindividually coupled to different capture oligonucleotides and thenbound together to form a single article, such as a matrix.

Typically, the support (whether a single or particulate support) iscapable of binding or otherwise holding the capture oligonucleotide tothe surface of the support in a sufficiently stable manner to accomplishthe purposes described herein. Such binding can include, for example,the formation of covalent, ionic, coordinative, hydrogen, or van derWaals bonds between the support and the capture oligonucleotides orattraction to a positively or negatively charged support. Captureoligonucleotides are attached to the solid support surface directly orvia linkers. In one embodiment, capture oligonucleotides are directlyattached to the support surface by providing or derivatizing either thesurface, the oligonucleotide, or both, with one or more reactive groups.For example, the surface of the Luminex™ particles can be modified with,for example, carboxylate, maleimide, or hydrazide functionalities oravidin and glass surfaces can be treated with, for example, silane oraldehyde (to form Schiff base aldehyde-amine couplings with DNA). Insome embodiments, the support or a material disposed on the support (as,for example, a coating on the support) includes reactive functionalgroups that can couple with a reactive functional group on the captureoligonucleotides. As examples, the support can be functionalized (e.g.,a metal or polymer surface that is reactively functionalized) or containfunctionalities (e.g., a polymer with pending functional groups) toprovide sites for coupling the capture oligonucleotides.

As an alternative, the capture oligonucleotides can be retained on thesurface by cross-linking of the capture oligonucleotides. Preferably, acapture oligonucleotide that is cross-linked includes a cross-linkingportion and a capture portion, where the capture portion includes amolecular recognition sequence that hybridizes to the tagging sequenceof the target oligonucleotide.

As yet another alternative, the support can be partially or completelycoated with a binding agent, such as streptavidin, antibody, antigen,enzyme, enzyme cofactor or inhibitor, hormone, or hormone receptor. Thebinding agent is typically a biological or synthetic molecule that hashigh affinity for another molecule or macromolecule, through covalent ornon-covalent bonding. The capture oligonucleotide is coupled to acomplement of the binding agent (e.g., biotin, antigen, antibody, enzymecofactor or inhibitor, enzyme, hormone receptor, or hormone). Thecapture oligonucleotide is then brought in contact with the bindingagent to hold the capture oligonucleotide on the support. Other knowncoupling techniques can be readily adapted and used in the systems andmethods described herein.

Capture and Target Oligonucleotides

The capture oligonucleotide includes a molecular recognition sequencethat can capture, by hybridization, a target oligonucleotide having acomplementary tagging sequence. The hybridization of the molecularrecognition sequence of a capture oligonucleotide and the taggingsequence of a target oligonucleotide results in the coupling of thetarget oligonucleotide to the solid support. The molecular recognitionsequence and tagging sequence are associated with a particularanalyte-specific sequence (also part of the target oligonucleotide),thus indicating, if hybridization occurs, the presence or concentrationof analyte with the analyte-specific sequence (or its complement) in theoriginal sample.

The coding and tagging sequences typically include at least sixnucleotides and, in some instances, include at least 8, 10, 15, or 20 ormore nucleotides. In some assays, as described below, the molecularrecognition sequence and tagging sequence include one or morenon-standard bases. In other assays, the molecular recognition sequenceand tagging sequence do not contain non-standard bases.

The capture oligonucleotide also typically includes a functional groupthat permits binding of the capture oligonucleotide to the solid supportor functional groups disposed on or extending from the solid support.The functional group can be attached directly to the polymeric backboneor can be attached to a base in the nucleotidic sequence. As analternative, the capture oligonucleotide can include a crosslinkingportion to facilitate crosslinking, as described above, or can beelectrostatically held on the surface. The capture oligonucleotides canbe formed by a variety of techniques, including, for example, solidstate synthesis, DNA replication, reverse transcription, restrictiondigest, run-off transcription, and the like.

In addition to the tagging sequence, the target oligonucleotide includesan analyte-specific sequence which corresponds to or is a complement toa sequence of interest in the analyte. The analyte-specific sequence canbe independent from the tagging sequence or some or all of the taggingsequence can be part of the analyte-specific sequence.

The length of the capture oligonucleotides can be optimized for desiredhybridization strength and kinetics. Usually, the length of themolecular recognition sequence is in the 6 to 20 (preferably, 8 to 12)nucleotide range. In a preferred embodiment, the different molecularrecognition sequences of the capture oligonucleotides are notcomplementary to one another and, more preferably, to any known naturalgene sequence or gene fragment that has a significant probability ofbeing present in a substantial amount in the sample to be tested. As aresult, the capture molecular recognition sequences of the captureoligonucleotides will primarily hybridize to the respectivecomplementary tagging sequences of the target oligonucleotides.

The target oligonucleotide (or an oligonucleotide complementary to atleast a portion of the target oligonucleotide) includes a reporter or acoupling agent for attachment of a reporter. The reporter or couplingagent can be attached to the polymeric backbone or any of the bases ofthe target or complementary oligonucleotide. Techniques are known forattaching a reporter group to nucleotide bases (both natural andnon-standard bases). Examples of reporter groups include biotin,digoxigenin, spin-label groups, radio labels, DNA-cleaving moieties,chromaphores, and fluorophores such as fluoroscein. Examples of couplingagents include biotin or substituents containing reactive functionalgroups. The reporter group is then attached to streptavidin or containsa reactive functional group that interacts with the coupling agent tobind the reporter group to the target or complementary oligonucleotide.

Polymerase Chain Reaction (PCR) Techniques

A variety of Polymerase Chain Reaction (PCR) techniques are known andcan be used in the assays described below. PCR techniques are typicallyused for the amplification of at least a portion of an oligonucleotide.The sample to be tested for the presence of an analyte-specific sequenceis contacted with the first and second oligonucleotide primers; anucleic acid polymerase; and nucleotide triphosphates corresponding tothe nucleotides to be added during PCR. The natural base nucleotidetriphosphates include dATP, dCTP, dGTP, dTTP, and dUTP. Nucleosidetriphosphates of non-standard bases can also be added, if desired orneeded. Suitable polymerases for PCR are known and include, for example,thermostable polymerases such as native and altered polymerases ofThermus species, including, but not limited to Thermus aquaticus (Taq),Thermus flavus (Tfl), and Thermus thermophilus (Tth), as well as theKlenow fragment of DNA polymerase I and the HIV-1 polymerase.

The first and second primers are complementary to different portions ondifferent strands of the double stranded oligonucleotide that is to beamplified. The sequence of the oligonucleotide that is amplifiedincludes the two primer sequences that hybridize to the analyte and theregion between the two primers. The primers can be formed by a varietyof techniques including, for example, solid state synthesis, DNAreplication, reverse transcription, restriction digest, run-offtranscription, and the like.

PCR includes the cycling steps of (i) annealing the firstoligonucleotide primer and the second oligonucleotide primer to thedouble stranded oligonucleotide that is to be amplified or to extensionproducts formed in previous cycles; (ii) extending the annealed firstand second oligonucleotide primers by the nucleic acid polymerase tosynthesize primer extension products; and (iii) denaturing the productsto obtain single stranded nucleic acids. Varieties of PCR have beendeveloped by modifying the steps or varying conditions (e.g., time andtemperature). Generally, any of these varieties of PCR can be used inthe assays described below, although some may be more useful than othersfor particular assays.

One variety of PCR developed for some of the assays described below is“fast-shot PCR” in which primer extension times are reduced oreliminated. As used herein, the term “fast-shot polymerase chainreaction” or “fast-shot PCR” refers to PCR where the extension stop, aswell as the stops for the annealing and melting steps, are very short oreliminated. Typically, for this method, the 3′ ends of the two primersare separated by no more than 10 bases on the template nucleic acid.

Enhanced specificity is achieved by using fast-shot PCR cycles where theextension stop, as well as the stops for the annealing and meltingsteps, are very short or eliminated. In some embodiments, the PCRsolution is rapidly cycled between about 90 to 100° C. and about 55 to65° C. with a maximum of about a one second hold at each temperature,thereby leaving the polymerase very little time to extend mismatchedprimers. In one embodiment, the reaction is cycled between about 95° C.and about 58° C. with about a one second hold at each temperature.

This rapid cycling is facilitated by generating a short PCR product by,in general, leaving a gap of about zero (0) to ten (10) bases on thetemplate nucleic acid between the 3′ bases of the first and secondprimers. Preferably, the primers are designed to have a Tm ofapproximately 55 to 60° C. For some embodiments, a total of about 37cycles is typically adequate to detect as little as 30 targetoligonucleotides.

Allele specific PCR primers can be used to discriminate SNP (singlenucleotide polymorphism) and other alleles. For SNP detection, theseprimers are designed to be complementary to each allele such that thepolymorphic base of interest is positioned at or near (typically, withinthree or five bases) the 3′ end of the first or second primer. Highlevels of allelic discrimination are achieved in part by the limitedability of Taq polymerase to extend a primer which has a nucleotidemismatch at its 3′ end with that of the target DNA, i.e., thecorresponding allele to which the primer is not specific. Otherpolymerases can also be used.

Additionally, allelic discrimination can be obtained by placing themismatch at other positions in the allele specific primer. Thesealternate positions for the nucleotide mismatch in the primer can beused to achieve selective amplification in two primary ways: 1) bysimply lowering the Tm (melting temperature) of the primer so that it isnot hybridized on the template DNA during thermal cycling so that thepolymerase can not extend the primers, or 2) by creating an unfavorableprimer/template structure that the polymerase will not extend.

Examples of Assays

Assays with Non-standard Bases in the Coding and Tagging Sequences

In one assay illustrated in FIG. 4, two or more groups of captureoligonucleotides 202 are prepared. Each group of captureoligonucleotides 202 includes a unique molecular recognition sequence204. The molecular recognition sequence of each group includes at leastone (and, typically, two or more) non-standard bases (denoted by the useof dashed lines in the Figures). The use of non-standard basessubstantially reduces the likelihood that the capture oligonucleotideswill hybridize with sequences that include only natural bases. This willtypically result in less non-specific hybridization when compared to asimilar assay using oligonucleotides with only natural bases. Thecapture oligonucleotide also typically includes a reactive functionalgroup for attachment to the solid support 206, although other attachmentmethods can be used, as described above.

The support for the assay can be, for example, a single solid support,such as, for example, a glass, metal, plastic, or inorganic chip. Thecapture oligonucleotides are disposed on the support and typically heldby one of the methods described above (e.g., coupling via reactivegroups on the capture oligonucleotide and support, use of a bindingagent disposed on the support, or cross-linking of the captureoligonucleotides). Each of the groups is disposed in one or more uniqueregions of the solid support so that the region(s) can be associatedwith a particular capture oligonucleotide.

In another embodiment (not shown), the support for the assay is aparticulate support (e.g., beads). It will be understood that any of theassays described herein can be performed on a single solid support, on aparticulate support, or any other support. The particulate support isdivided into groups of particles, each group of particles having acharacteristic (e.g., color, shape, size, density, or other chemical orphysical property) that distinguishes that group of particles from othergroups. Each group of capture oligonucleotides is coupled to one or moregroups of particles. This produces an association of a particular groupof particles with a particular group of capture oligonucleotides,allowing the determination of the capture oligonucleotide by observationof the unique particle support characteristic.

Returning to FIG. 4, the target oligonucleotide 208, if present in theassayed sample, contains an analyte-specific sequence 210 and a taggingsequence 212 complementary to the molecular recognition sequence 204 ofone group of the capture oligonucleotides 202. The tagging sequence 212contains at least one non-standard base; otherwise the tagging sequencewould not be complementary to the molecular recognition sequence of thecapture oligonucleotide. An oligonucleotide 214 complementary to aportion of the target oligonucleotide 208 includes a reporter 216 or acoupling agent (not shown) for attachment of a reporter.

The target oligonucleotide 208 and complementary oligonucleotide 214 canbe formed by, for example, PCR amplification of an analyte containingthe analyte-specific sequence or its complement. In PCR amplification,two different primers are used (as illustrated at B of FIG. 4). A firstprimer 218 contains a sequence complementary to a first sequence on afirst strand of the analyte 220. A second primer 222 contains a sequencethat is the complementary to a second sequence on a second strand of theanalyte 220 which is upstream or downstream of the first sequence. Theanalyte-specific sequence typically includes the sequence of the analytestretching between, and including, the sequences (or complements) towhich the primers hybridize. The first primer 218 includes the taggingsequence 212 and the second primer 222 includes the reporter 216 (or acoupling agent for a reporter). Extension of the first and secondprimers and amplification proceeds using known PCR amplificationtechniques or the fast-shot PCR techniques described above to producethe target oligonucleotide 208 and complementary oligonucleotide 214 (asillustrated at C of FIG. 4). Other known synthetic methods, such as, forexample, solid state synthesis, DNA replication, reverse transcriptionand the like, can be used to form the target and complementaryoligonucleotides.

Returning to the assay, the target oligonucleotide 208 is typicallybrought into contact with the support 206 (or a container holding aparticulate support) with associated capture oligonucleotides 202.Conditions are controlled to promote selective hybridization of thetagging sequence of the target oligonucleotide with a complementarymolecular recognition sequence of a capture oligonucleotide, if anappropriate capture oligonucleotide is present on the support (asillustrated at D of FIG. 4). A reporter is also added (unless thecomplementary oligonucleotide 214 already contains the reporter) forcoupling to the complementary oligonucleotide 214. Optionally,unincorporated primers can be removed prior to hybridization bytechniques such as, for example, size exclusion chromatography,differential precipitation, spin columns, or filter columns, or afterhybridization by, for example, washing.

For assays on a planar solid support, the assay can be read bydetermining whether the reporter group is present at each of theindividual regions on the support. The presence of the reporter groupindicates that the original sample contains an analyte having theanalyte-specific sequence associated with the particular taggingsequence and molecular recognition sequence for that region of thesupport. The absence of the reporter group suggests that the sample didnot contain an analyte having the particular analyte-specific sequence.

For assays on particle supports, the particles can be separatedaccording to the unique characteristics and then it is determined whichparticles have a reporter coupled to the particle via the capture andtarget oligonucleotides. Techniques for accomplishing the separationinclude, for example, flow cytometry. The presence of the reporter groupindicates that the sample contains the target oligonucleotide having theanalyte-specific sequence associated with a particular tagging sequenceand the molecular recognition sequence of a particular captureoligonucleotide.

The assay illustrated in FIG. 4 can be adapted for use in determiningthe presence of alleles in a sample. For example, the assay includesallele-specific primers (either the first or second primers 218, 222 orboth) corresponding to two or more alleles. Each of the allele-specificprimers includes a sequence that specifically hybridizes to only oneallele. The tagging sequence or reporter (or coupling agent) attached tothe allele-specific primer is also specific for the allele. If theallele is present in the sample, the allele-specific primer(s)associated with that allele will extend and will be detected by eitherhybridizing to a complementary, allele-specific capture oligonucleotideon the support or observing an allele-specific reporter group. It willbe recognized that the assay can also be used to determine the presenceor absence of non-allelic analyte-specific sequences in the analyte.

This method can be used to detect SNP (single nucleotide polymorphism)alleles. Either the first or second primers will be SNP-specific.Typically, two (or more) different SNP-specific primers will be used inthe assay. Preferably, the SNP-specific primers will have the SNP sitepositioned at or near (e.g., within three or five bases) the extendableend of the primer. “Fast-shot PCR” techniques can be useful in this SNPassay because the short extension times will substantially reduce thelikelihood that non-complementary primers will extend.

Hybridization of the capture oligonucleotides and targetoligonucleotides is a feature of the assays described herein. Thishybridization takes place in a hybridization mixture that contains salts(e.g., sodium salts or magnesium salts), a buffer (e.g., TRIS, TAPS,BICINE, or MOPs), a non-specific blocking agent (e.g., SDS, BSA, orsheared genomic DNA), and a protecting agent (e.g., EDTA or an azide),as is used in many conventional hybridization methods. Typically, thehybridization takes place at a sodium ion (or other cation)concentration of at least 0.01 to 1.0 M and a pH of 7.0 to 8.3.Generally, this hybridization and any washing steps are performed at atemperature and salt concentration that meet desired stringencyconditions for maintaining hybridization. Stringency conditions aresequence dependent. Stepwise increases in stringency conditions can beused, if desired, over several washing steps.

“Low stringency conditions” are selected to be about 10 to 15° C. belowthe thermal melting point (Tm) for the specific sequence at the ionicstrength and pH of the hybridizing solution. Tm is the temperature (forthe ionic strength, pH, and nucleic acid concentration) at which about50% of the tagging sequences hybridize to complementary molecularrecognition sequences at equilibrium.

“Moderate stringency conditions” are selected to be about 5 to 10° C.below the thermal melting point (Tm) for the specific sequence at theionic strength and pH of the hybridizing solution.

“High stringency conditions” are selected to be no more than about 5° C.below the thermal melting point (Tm) for the specific sequence at theionic strength and pH of the hybridizing solution.

In another assay illustrated in FIG. 5, two or more groups of captureoligonucleotides 252 are prepared and placed on a support 256, asillustrated at A of FIG. 5. Each group of capture oligonucleotides 252includes a unique molecular recognition sequence 254. The molecularrecognition sequence of each group includes at least one (and,typically, two or more) non-standard bases. A target oligonucleotide 258and complementary oligonucleotide 264 can be formed by, for example, PCRamplification of an analyte containing the analyte-specific sequence orits complement. In PCR amplification, two different primers are used (asillustrated at B and C of FIG. 5). A first primer 268 contains asequence complementary to a first sequence on a first strand of theanalyte 270. A second primer 272 contains a sequence that is thecomplementary to a second sequence on a second strand of the analyte 270which is upstream or downstream of the first sequence. Theanalyte-specific sequence typically includes the sequence of the analytestretching between, and including, the sequences (or complements) towhich the primers hybridize. The first primer 268 includes the taggingsequence 262 and the second primer 272 includes the reporter 266 (or acoupling agent for a reporter).

The target oligonucleotide 258 is typically brought into contact withthe support 256 (or a container holding a particulate support) withassociated capture oligonucleotides 252. Conditions are controlled topromote selective hybridization of the tagging sequence of the targetoligonucleotide with a complementary molecular recognition sequence of acapture oligonucleotide, if an appropriate capture oligonucleotide ispresent on the support (as illustrated at D of FIG. 5). A reporter isalso added (unless the complementary oligonucleotide 264 alreadycontains the reporter) for coupling to the complementary oligonucleotide264. Optionally, unincorporated primers can be removed prior tohybridization by techniques such as, for example, size exclusionchromatography, or after hybridization by, for example, washing.

An enzyme 280 is then provided to covalently couple the complementaryoligonucleotide 264 to the capture oligonucleotide 252. Suitable enzymesinclude ligases. Optionally, the target oligonucleotide 258 is denaturedfrom the complementary oligonucleotide 264 and the targetoligonucleotide and other components of the assay are washed awayleaving the complementary oligonucleotide 264 bound to the support 256,as illustrated at E of FIG. 5. The reporter 266 can then be detected.

In yet another assay illustrated in FIG. 6, the target oligonucleotide314 forms a hairpin or stem-loop structure 321, 323 (or structure otherthan the typical double helix). In this assay, each of the first andsecond primers 318, 322 includes a portion of the tagging sequence 312 bor a complement to a portion of the tagging sequence 312 a. In addition,one of the primers 322 has a reporter 316 (or coupling agent for areporter) attached to the portion of the tagging sequence 312 b. Using,for example, PCR techniques, the first and second primers 318, 322amplify the analyte 320 to produce a target oligonucleotide 314 and itscomplement 308. The tagging sequence 312 b, 313 a of the targetoligonucleotide 314 is distributed at both ends of the targetoligonucleotide.

The target oligonucleotide 314 is denatured from its complement 308 andbrought into contact with the solid support 306 having captureoligonucleotides 302 with molecular recognition sequences 304. If themolecular recognition sequence 304 of one of the captureoligonucleotides is complementary to the tagging sequence 312 b, 313 aof the target oligonucleotide 314, the target oligonucleotide 314 willhybridize to that capture oligonucleotide. In some embodiments, thecapture oligonucleotide is divided into two parts, each partcomplementary with one of the parts of the tagging sequence 312 b, 313a. The two parts are coupled by a linker. The linker can be additionalnucleotides or any other chemical linking moiety. The target sequence ofthe target oligonucleotide 314 forms at least part of a stem-loopstructure 321, 323 (or structure other than an double helix). Detectionis then performed as discussed above in the previous example.

In an alternative assay illustrated in FIG. 7, an analyte 420 iscontacted by initial primers 440, 442 each having a sequence that iscomplementary to a sequence of the analyte 420, as illustrated at A ofFIG. 7. One of the initial primers 440 also includes a coupling group444 (e.g., biotin or a substituent containing a reactive functionality)for attachment to a substrate 450. The initial primers 440, 442 areextended using, for example, PCR techniques, as illustrated at B of FIG.7. The extended initial primers 446, 448 each include theanalyte-specific sequence or its complement.

The extended initial primers 446, 448 are then brought into contact witha substrate 450 that interacts with the coupling group 444 of extendedinitial primer 446 to attach the extended initial primer 446 to thesubstrate 450, as illustrated at C of FIG. 7. For example, the substratecan be coated with streptavidin and the extended initial primer includebiotin.

Next, first and second primers 418, 422 are brought into contact withthe extended initial primers 446, 448, as illustrated at C of FIG. 7.The first primer 418 has a tagging sequence 412 and the second primer422 has a reporter 416 (or coupling agent for a reporter). Both primersalso include a sequence complementary to a section of the extendedinitial primers 446, 448. The assay illustrated in FIG. 7 also showsthat other primers 422 a can be added. This is not a necessary featureof the assay, but is used to illustrate one embodiment of an assay fordetecting alleles. The use of allele specific primers can be used in anyof the other assays illustrated herein.

In the illustrated assay, primers 422, 422 a are allele-specific primerswith allele-specific reporters 416, 416 a. In the illustrated example,the alleles differ by a single nucleotide, although it will beunderstood that other allele-specific assays with more than onenucleotide difference can be performed using these techniques. Primer422 is extended because it is complementary to a sequence on theextended initial primer 446. Primer 422 a does not extend because it isnot complementary to extended initial primer 446. It will be recognizedthat an alternative assay includes several different allele-specificprimers with allele-specific tagging sequences (as opposed toallele-specific reporters). It will also be recognized that anotheralternative assay includes non-allelic primers for determination of thepresence of absence of non-allelic analyte-specific sequences in theanalyte.

The primers 418, 422 are extended to form the target oligonucleotide 408with the tagging sequence 412 and the complementary oligonucleotide 414with the reporter 416 (or a coupling agent for a reporter). The targetoligonucleotide 408 and complementary oligonucleotide 414 are denaturedfrom the extended initial primers 446, 448 and brought into contact withcapture oligonucleotides 402 on a solid support 406 (e.g., chip, wafer,or particles). The target oligonucleotide 414 hybridizes to a captureoligonucleotide 402 having a molecular recognition sequence 404complementary to the tagging sequence 412. The presence or absence ofparticular analyte-specific sequences in the analyte is determined byobservation of the presence or absence of reporter associated with eachunique group of capture oligonucleotides.

In another example of an assay, a first primer 468 and a second primer472 are brought into contact with an analyte 470 and extended to form atarget oligonucleotide 458 and complementary oligonucleotide 464. In theillustrated example, the first and second primers 468, 472 are bothallele-specific, but specific to different alleles. In addition to thefirst and second primers 468, 472, other first and second primers 469,473 are included to amplify other alleles, if present in the sample.

The first primer 468 includes a first part 462 a of a tagging sequenceand the second primer 472 includes a second part 462 b of the taggingsequence. One of the parts 462 a, 462 b includes a reporter 466 (orcoupling agent for a reporter). Typically, the parts 462 a, 462 b of thetagging sequence will be configured so that the extension of the primers468, 472 does not proceed through the tagging sequence. For example, theparts 462 a, 462 b can include a non-standard base as the base linkingthe part of the tagging sequence to the extendable portion of theprimers 468, 472. In this embodiment, the nucleotide triphosphate of thecomplement of the non-standard base is not included in the PCRamplification process. Alternatively, a chemical linker can be used tocouple the part of the tagging sequence to the extendable portion of theprimer. Examples of suitable linkers include, but are not limited to,n-propyl, triethylene glycol, hexaethylene glycol, 1′,2′ dideoxyribose,2′-O-methylriboneucleotides, deoxyisocytidine, or any linkage that wouldhalt the polymerase.

A coupling oligonucleotide 452 is provided on a support 456. Thecoupling oligonucleotide 452 includes parts 453 a, 453 b that arecomplementary to the parts 462 a, 462 b of the tagging sequence. Theseparts 453 a, 453 b are coupled by a chemical or nucleotidic linker 454that is capable of coupling 5′ (or 3′) ends of two nucleotidicsequences.

The target oligonucleotide 458 and complementary oligonucleotide 464 arebrought in contact with the support 456 and capture oligonucleotide 452to hybridize the corresponding parts 453 a, 453 b of the captureoligonucleotide with the respective parts 462 a, 462 b of the taggingsequence. The remainder of the target oligonucleotide 458 andcomplementary oligonucleotide 464 will typically form a structure suchas that illustrated in FIG. 8.

Assays in which Non-standard Bases are Added by PCR Techniques

Although labeled natural nucleotide bases have many uses, there areshortcomings associated with labeled natural nucleotides. For example,site specific incorporation of a labeled natural nucleotide base isdifficult to achieve. Generally, to label a position in anoligonucleotide which contains adenine, labeled adenosine triphosphate(dATP*) is added as a substrate to a reaction mix which includes anoligonucleotide template, dGTP, dCTP and dTTP, and a polymerase enzyme.If all dATP's in the reaction mix are labeled, all the adenine residuesin the oligonucleotide sequence will be labeled. If a fraction of thedATP's in the reaction mix are labeled, adenine residues in randompositions in the sequence are labeled. It is thus extremely difficult tolabel a single nucleotide residue in an oligonucleotide.

To overcome the problems associated with the incorporation of multiplelabeled nucleotide residues, labeled dideoxyribonucleic acids have beenused. Because the dideoxyribonucleic acid lacks a 3′ hydroxyl group, theoligonucleotide is terminated at the position where the labeleddideoxyribonucleic acid is introduced. To determine the position of thelabeled nucleotide, ladders are run to sequence the oligonucleotide.Because the oligonucleotide is terminated at the position where thedideoxyribonucleic acid is introduced, dideoxyribonucleic acids cannotgenerally be used in connection with amplification of theoligonucleotide strand.

FIG. 9 illustrates one type of assay, according to the invention, whichincludes the incorporation of a non-standard base by PCR. First andsecond primers 518, 522 are hybridized to analyte 520 and extended. Oneof the primers 522 includes a non-standard base 550 which, whenextended, becomes the target oligonucleotide 508. Optionally, additionalbases can be provided after the non-standard base 550. The targetoligonucleotide 508 with the non-standard base 550 is then brought intocontact with the solid support 506 a, 506 b that includes captureoligonucleotides 502 a, 502 b. The solid support illustrated in FIG. 9is the particulate support discussed above, however, it will berecognized that a single solid support (e.g., a chip or wafer) couldalso be used.

The capture oligonucleotides 502 a, 502 b are different and are attachedto different supports 506 a, 506 b, respectively, so that the captureoligonucleotide can be recognized by observing the unique property ofthe support to which it is attached. One capture oligonucleotide 502 ahybridizes with the target oligonucleotide 508. The captureoligonucleotide 502 a in this embodiment has a sequence that iscomplementary to at least a portion of the analyte-specific sequence ofthe target oligonucleotide 508.

After hybridization of the target oligonucleotide 508, the captureoligonucleotide 502 a is extended in a PCR solution that includes dATP,dUTP, dGTP, dCTP, and the nucleotide triphosphate of a secondnon-standard base (e.g., diso-GTP) 552 complementary to the non-standardbase 550 on the target oligonucleotide 508. The second non-standard base552 is labeled with a reporter 516 (or coupling agent for a reporter).As the capture oligonucleotide is extended, the second non-standard base552 with the reporter 516 is incorporated into the extended captureoligonucleotide opposite the non-standard base 550. Thus, the presenceor absence of a reporter on a particular group of particulate supportsindicates the presence or absence of a particular target oligonucleotideassociated with the capture oligonucleotide.

FIG. 10 illustrates another assay. In this assay, the first primer 618includes a tagging sequence 612 and the second primer 622 has anon-standard base 621 (or a sequence containing a non-standard base) atits 5′ end. The primers 618, 622 amplify the analyte 620 in the presenceof the dATP, dCTP, dGTP, dTTP, and the nucleotide triphosphate of thenon-standard base complementary to non-standard base 621. Thisnon-standard base nucleotide triphosphate is labeled with a reporter 616(or coupling group for a reporter) and is incorporated oppositenon-standard base 621 to form the target oligonucleotide 608.

The target oligonucleotide 608 is brought into contact with the solidsupport 606 having capture oligonucleotides 602 with molecularrecognition sequences. If one of the molecular recognition sequences iscomplementary to the tagging sequence 612 of the target oligonucleotide608, the target oligonucleotide 608 will hybridize to the captureoligonucleotide 602. Detection is then performed as discussed above inthe previous examples.

FIG. 11 illustrates yet another assay. In this assay, the first primer718 includes a tagging sequence 712 and the second primer 722 has anon-standard base 721 followed by a natural base 723 (or a sequence ofnatural bases) at its 5′ end. The primers 718, 722 amplify the analyte720 in the presence of the dATP, dCTP, dGTP, and dTTP only to form apartially extended target oligonucleotide 707 and its complement 714.The extension of the partially extended target oligonucleotide islimited by the non-standard base 721. After this initial amplification,the amplification products 707, 714 are washed to remove dATP, dCTP,dGTP, and dTTP.

A second extension step is then performed, in the presence of thetriphosphate of the non-standard base complementary to non-standard base721 and at least the triphosphate of the natural base complementary tonatural base 723. This natural base triphosphate is labeled with areporter 716 (or coupling group for a reporter) and is incorporatedopposite natural base 723 to form the target oligonucleotide 708.

The target oligonucleotide 708 is brought into contact with the solidsupport 706 having capture oligonucleotides 702 with molecularrecognition sequences. If one of the molecular recognition sequences iscomplementary to the tagging sequence 712 of the target oligonucleotide708, the target oligonucleotide 708 will hybridize to the captureoligonucleotide 702. Detection is then performed as discussed above inthe previous examples.

In one embodiment, allele-specific second primers are used with the samefirst primer. The allele-specific second primers are differentiated inthe portion of the second primer that anneals to the analyte. Adifferent natural base 723 is selected for each allele. During thesecond extension step, where bases are added opposite the non-standardbase 721 and natural base 723, the nucleotide triphosphates of two ormore natural bases are added to the extension mixture. The differentnucleotide triphosphates are labeled with different reporters. Thus, ifthe natural base 723 can be A or C, depending on the allele, the dTTPand dGTP used in the extension step are labeled with differentreporters. The identity of the reporter can be used to determine thepresence of a particular, associated allele. Thus, for example, fourdifferent alleles can be simultaneously tested using this method and,with appropriate choice of reporters, can be indicated using fourdifferent colors.

Other Assays

In one assay illustrated in FIG. 16, two or more groups of captureoligonucleotides 902 are prepared. Each group of captureoligonucleotides 902 includes a unique molecular recognition sequence904. The molecular recognition sequence of each group optionallyincludes at least one or more non-standard bases. The captureoligonucleotide also typically includes a reactive functional group forattachment to a solid support 906, although other attachment methods canbe used, as described above.

In one embodiment, the support for the assay is a particulate support(e.g., beads). It will be understood that any of the assays describedherein can be performed on a single solid support, on a particulatesupport, or any other support. The particulate support is divided intogroups of particles, each group of particles having a characteristic(e.g., color, shape, size, density, or other chemical or physicalproperty) that distinguishes that group of particles from other groups.Each group of capture oligonucleotides is coupled to one or more groupsof particles. This produces an association of a particular group ofparticles with a particular group of capture oligonucleotides, allowingthe determination of the capture oligonucleotide by observation of theunique particle support characteristic.

In another embodiment (not shown), the support for the assay can be, forexample, a single solid support, such as, for example, a glass, metal,plastic, or inorganic chip. The capture oligonucleotides are disposed onthe support and typically held by one of the methods described above(e.g., coupling via reactive groups on the capture oligonucleotide andsupport, use of a binding agent disposed on the support, orcross-linking of the capture oligonucleotides). Each of the groups isdisposed in one or more unique regions of the solid support so that theregion(s) can be associated with a particular capture oligonucleotide.

Returning to FIG. 16, the target oligonucleotide 908, if present in theassayed sample, is contacted with a first primer 909 and a second primer911. The first and second primers 909, 911 can be allele-specific or,preferably, are not complementary to allele specific portions of thetarget oligonucleotide (i.e., the allele specific portions of interestare positioned within the target oligonucleotide between the regionsthat hybridize to the two primers). The second primer 911 also includesa non-complementary attachment region 905. This non-complementaryreporter attachment region 905 optionally includes one or morenon-standard bases. The target oligonucleotide 908 is amplified usingthe first and second primers 909, 911 and PCR techniques to obtain anamplification product 907 that includes the reporter attachment region905.

The amplification product 907 is then contacted with allele specificprimers 920 a, 920 b that are then extended, if the particular allele ispresent, using reaction conditions and reaction components similar toPCR to provide an allele specific extension product 922. Each allelespecific primer 920 a, 920 b has an allele-specific tagging sequence 912a, 912 b that is complementary to different molecular recognitionsequences 904 and capture oligonucleotides 902. When extending theallele specific primers 920 a, 920 b, a labeled nucleotide 925 (oroligonucleotide) that is complementary to one or more bases of theattachment region 905 is provided. The labeled nucleotide 925 oroligonucleotide can include a reporter or a coupling agent, such asbiotin, for attachment of a reporter.

After forming the extension product 922, contact is made with thecapture oligonucleotides 902 and with a reporter 930 (unless a reporterwas already attached). The capture oligonucleotide 902 and the support906 identify which allele(s) is/are present in the sample and thereporter provides for detection of the extension product 922. For assayson particle supports, the particles can be separated according to theunique characteristics and then it is determined which particles 906have a reporter coupled to the particle via the capture oligonucleotide902 and extension product 922. Techniques for accomplishing theseparation include, for example, flow cytometry. The presence of thereporter group indicates that the sample contains the allele associatedwith a particular allele-specific tagging sequence.

Selection of Molecular Recognition Sequences

When multiple molecular recognition sequences are used to form an assaysystem that can detect more than one analyte-specific sequence withapplication of a single sample, a collection of different molecularrecognition sequences is typically needed. Preferably, the molecularrecognition sequences are sufficiently different to permit reliabledetection of analyte-specific sequences under a desired set ofstringency conditions. A variety of different methods can be used tochoose the collection of molecular recognition sequences. The followingis a description of some methods and criteria that can be used. Themethods and criteria can be used individually or in combinations.

The following are examples of criteria that can be used in creating acollection of molecular recognition sequences: the number of bases inthe sequence, the number of non-standard bases in the sequence, thenumber of consecutive natural bases in the sequence, the number ofconsecutive bases (in either the forward or reverse directions) that arethe same in any two sequences, specific required sequences (e.g., GCclamps at the 3′ or 5′ ends or both) and the estimated or actual meltingtemperature. One example of a method for determining Tm is described inPeyret et al., Biochemistry, 38, 3468-77 (1999), incorporated herein byreference. The non-standard bases can be estimated or accounted forusing, for example, values for other bases (e.g., iso-G/iso-C can beestimated using G/C) or using experimental data such as that describedbelow.

The following are a set of steps that can be used to form the collectionof molecular recognition sequences:

1) Create a set of all possible oligonucleotides having a length of n₁(e.g., 8, 9, or 10 nucleotides) using the natural bases and the desirednon-standard bases (e.g., iso-C, iso-G, or both).

2) Optionally require that the oligonucleotides have a particularsubsequence (e.g., GC clamps on the 3′ or 5′ ends or both ends).

3) Remove oligonucleotides without at least n₂ non-standard bases (e.g.,without at least two iso-C bases) or with more than n₃ non-standardbases (e.g., with more than two iso-C bases) or both (e.g., accept onlyoligonucleotides with exactly two iso-C bases).

4) Optionally remove oligonucleotides with n₄ (e.g., four or five)natural bases in a row.

5) Select one of the remaining oligonucleotides and eliminate any of theremaining oligonucleotides that have n₅ bases (e.g., five or six bases)in the same order anywhere in the oligonucleotide sequence. Repeat foreach non-eliminated oligonucleotide.

6) Optionally select one of the remaining oligonucleotides and determineits reverse complement (e.g., the reverse complement of “ACT” is “AGT”),then eliminate any of the other oligonucleotides that have n₆consecutive bases (e.g., four or five bases) that are the same as aportion of the sequence of the reverse complement. Repeat for eachnon-eliminated oligonucleotide.

7) Optionally select only the remaining oligonucleotides that have anestimated or actual melting temperature (Tm) within a desiredtemperature range, above a desired temperature limit, or below a desiredtemperature limit For example, oligonucleotides can be eliminated thathaving a melting temperature below room temperature (about 22° C.).

EXAMPLES Example 1 Cross-hybridization Analysis of Coding and TaggingSequences

The equipment used in this analysis includes Luminex® 100 and Luminex®microbeads, DNA synthesizer (Northwestern Engineering, Inc.),Spectrophotometer for spot checking synthesis yields, thin layerchromatography (TLC) (SI250F TLC plate-silica gel, JTBaker) foroligonucleotide quality control, centrifuge, sonicator (Ney Dental),Vortex Genie (Vortex), and various pipettes (2, 20, 200, and 1000 μL).

A set of more than 100 oligonucleotides (molecular recognitionsequences) and their complements (tagging sequences) were designed andsynthesized. The two sets of oligonucleotides contained non-standard(isoC and isoG)(EraGen Biosciences, Inc., Madison, Wis.) and natural (A,G, C, and T) (Perkin-Elmer/ABI) nucleotides and were 9 to 10 bases inlength. The first set of the oligonucleotides was designated asmolecular recognition sequences and labeled on the five prime end withan amino modifier (C6-TFA, Glen Research). The complement sets ofoligonucleotides were designated the tagging sequence and labeled on thefive prime end with Cy3 (Glen Research).

The following reagents were used in coupling the molecular recognitionsequence to the unique Luminex beads: 0.1 mM pH4.5,2-[N-morpholino]ethanesulfonic acid (MES) (Sigma).

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC) (Pierce), 0.02%(v/v) Tween (Sigma), 0.1% (w/v) SDS (Sigma).

The hybridization step included a hybridization buffer Sourav 0.5containing 10 mM Tris (Sigma), 1 mM EDTA (Sigma), 200 mM NaCl (Aldrich),10 mM MgCl₂ (Aldrich), and 1% (w/v) PEG 8000 (Sigma).

Ninety-eight of the molecular recognition sequences were diluted to 1nmol/μL in MES. Ninety-eight unique sets of Luminex® beads were preparedfor coupling. The beads were sonicated for 20 seconds and vortexed for10 seconds before being aliquoted. From the stock beads (1.25×10⁷beads/mL), 5 million beads were selected and placed in a 1.5 mLmicrocentrifuge tube. The beads were centrifuged at 10,000 rcf for 1minute. The beads were then decanted, being careful not to disturb thebeads. Finally, the beads were brought to 50 μL in MES, sonicated andvortexed. To couple the molecular recognition sequence to a distinctbead, 1 nmol of each molecular recognition sequence was added to one ofthe unique bead sets. Next, 1.75 μL of a fresh EDC (20 mg EDC/1 mLddH2O) was added to the mixture, sonicated and vortexed. The mixture wasthen allowed to incubate at room temperature in the dark for 30 minutes,vortexing every 10 minutes. After 30 minutes, another 1.75 μL of a freshEDC was added and incubated for 30 minutes, vortexing every 10 minutes.

After coupling, the beads were washed by adding 400 μL Tween-20,vortexed, centrifuged (10,000 rcf/1 min) and decanted. Next 400 μL SDSwas added, centrifuged, decanted and finally brought up in 100 μL in MESand enumerated.

The complementary oligonucleotides (the tagging sequences) werequantified and qualified using TLC and polyacrylamide gel, and dilutedto a final working concentration of 50 fmol/μL in MOPS.

After enumeration, the Luminex® bead/molecular recognition sequenceswere combined into a 98 bead set (1000 beads/bead region/well) foranalysis. From the 98 bead set, a 50 bead set (2500 beads/beadregion/well) was created. Table 1 includes the molecular recognitionsequences for the 50 bead set and Table 2 includes the molecularrecognition sequences for the 98 bead set.

To setup the cross hybridization experiment, 50 femtomoles of taggingsequences (1->98) were pipetted into wells in two 96 well plates (wells1 and 2 were used for controls). Current limitations of the Luminex®100, trimmed the dataset to 98 tagging sequences, with 2 controls forbackground subtraction (no tagging sequence).

The master mix of beads (98 mix), 10 μL/well, was then added to eachwell along with 31 μL of 2× Sourav 0.5 hybridization buffer andsufficient quantity of ddH2O, to give a final volume of 62 μL/well. Thereagents were mixed well and allowed to incubate at room temperature forapproximately 10 minutes. The samples were immediately analyzed by flowcytometry on the Luminex®100.

The 50 bead master mix was also run with its complementary molecularrecognition sequences and tagging sequences, however the taggingsequences were at 500 fmol per well.

The resulting data is reported as Median Fluorescence Intensity (MFI)per bead for both sets. FIG. 12 shows the 3D surface map graphicalresults of the data collected in the 98 bead master mix experiment. TheY axis represents the molecular recognition sequence and the X axisrepresents the tagging sequence. FIG. 13 shows the 3D surface mapgraphical results of the data collected in the 50 bead master mixexperiment.

TABLE 1 50 Bead Molecular recognition  sequences (Y = iso-G and  X =iso-C) Molecular recognition Bead No. sequence Seq Id No: 1 GAXGTXTGTC 12 CXGTTXTTCC 2 3 GGXTTGXTAG 3 4 C1TXGXTCTC 4 5 CXTCAXGAAC 5 6 GTAGXTAXGC6 7 GGAXGXTAAC 7 8 CXGTATXGTG 8 9 CATXGGTAXG 9 10 GATTXTCGXC 10 11GTTXAXGACC 11 12 CXGAAXGATC 12 13 CAAXTACGXC 13 14 CGGXATAXAC 14 15GXAAAXXAGG 15 16 GTCXTAGXXC 16 17 GXCCTXTAXC 17 18 CCXACXTGAG 18 19CTXXCAXAGG 19 20 GTXGAXATGC 20 21 GAAAXTGXXG 21 22 GCTGXAXATC 22 23CGCAXATXAC 23 24 CTGGXTCXAG 24 25 GGAAXAXXCC 25 26 CXTCGCXTAC 26 27GXCXAAAAXG 27 28 CXXGACXATC 28 29 CCATXAGXCC 29 30 GGCAXTXTGG 30 31CTXAACXGGG 31 32 GGAXACGXG 32 33 GCGXTTTAXG 33 34 GAGXAGXTXC 34 35GXCTAAXCCG 35 36 GCXTGTXCAC 36 37 GXCAGAXTCG 37 38 CGTXCTAGXG 38 39CGXXTAGTXG 39 40 CXAGGXAACC 40 41 CXAGAXGAXG 41 42 CGXTGXGTC 42 43CAGXCGTXAG 43 44 GGCTXTGXAC 44 45 CCAGXGXAAG 45 46 GGCXAATXGC 46 47GXCTGCXGG 47 48 GAXCTXCGGC 48 49 GTXCGAXGGG 49 50 GGXXATCCXG 50

TABLE 2 98 Bead Molecular recognition  sequences (Y = iso-G and  X =iso-C) Molecular recognition Bead No. sequence Seq Id No: 1 GAXGTXTGTC 12 CXGTTXTTCC 2 3 GGXTTGXTAG 3 4 CTTXGXTCTC 4 5 CXTCAXGAAC 5 6 GXCTTCXATG51 7 GTAGXTAXGC 6 8 GGAXGXTAAC 7 9 CXGTATXGTG 8 10 CATXGGTAXG 9 11GATTXTCGXC 10 12 GTTXAXGACC 11 13 CXTCTTXXCC 52 14 CXGAAXGATC 12 15CAAXTACGXC 13 16 CTCTXAXCCC 53 17 CTCXTGGTXC 54 18 CGGXATAXAC 14 19GXAAAXXAGG 15 20 GTCXTAGXXC 16 21 GXCCTXTAXC 17 22 CCXACXTGAG 18 23CTXXCAXAGG 19 24 GXCAAAXCAC 55 25 GTXGAXATGC 20 26 GTTXGCXTTG 56 27GAAAXTGXXG 21 28 GCTGXAXATC 22 29 CXCXTXCAAC 57 30 CTXXACAXXC 58 31CXACTCXACC 59 32 GACXCAXXTG 60 33 CGCAXATXAC 23 34 CTCXCTXACG 61 35CTGGXTCXAG 24 36 GGAAXAXXCC 25 37 GTGGXCTXTC 62 38 CXTCGCXTAC 26 39CAXXACCXAG 63 40 GXCXAAAAXG 27 41 GTXCXAXACC 64 42 CXXGACXATC 28 43CCATXAGXCC 29 44 CACXXTGXTC 65 45 GGCAXTXTGG 30 46 CTXAACXGGG 31 47GXTCCTXGTC 66 48 GGAXACGXG 32 49 GCGXTTTAXG 33 50 CCXXATGTXG 67 51GAGXAGXTXC 34 52 GXCTAAXCCG 35 53 GCXTGTXCAC 36 54 GXCAGAXTCG 37 55CGTXCTAGXG 38 56 CGXXTAGTXG 39 57 CXAGGXAACC 40 58 GXGGTTXXTC 68 59CXAGAXGAXG 41 60 CGXTGXGTC 42 61 CAGXCGTXAG 43 62 GGCTXTGXAC 44 63CXCCGXAATC 69 64 GXXACXACAC 70 65 GCXCXGTXC 71 66 GXCXGGAXC 72 67CGAXAGCAXC 73 68 CCCAXTCCXC 74 69 GTXCCXXCAG 75 70 CXCCTAXCGG 76 71GXGTTGXCG 77 72 CXAAGXAXCG 78 73 GGAGXCXXTC 79 74 CXGXAXGTAC 80 75GXACGAXTXG 81 76 GXGCTXCATG 82 77 GTGXAGAGXG 83 78 GCCGXCXTC 84 79CAAXCGXTCG 85 80 CACAXACXGC 86 81 CCAGXGXAAG 45 82 GGCXAATXGC 46 83GXCTGCXGG 47 84 GXTGGXXCG 87 85 GCCXCCXGT 88 86 CXAXGGTCXC 89 87CCXXGXGTG 90 88 GGXACXCCAG 91 89 GAXCTXCGGC 48 90 GCCTXCXGAC 92 91GTXCGAXGGG 49 92 CXTTXCGCXC 93 93 GGXXATCCXG 50 94 CXCTAXGXXG 94 95CXGCXAGXG 95 96 CXAGCXACGG 96 97 GACAXGCXCC 97 98 GGGXCGXXA 98

Example 2 Preliminary Determination of Non-standard Base Contributionsto the Nearest-Neighbor Parameters for Predicting Nucleic Acid DuplexStability

A Beckman DU-7500 spectrometer with temperature controller and samplecarriage was utilized. Six samples can simultaneously be measured withprecise temperature control. In order to cover a one hundred fold rangeof sample concentrations, quartz cuvettes of pathlengths 0.1 cm, 0.2 cm,0.5 cm and 1.0 cm, were obtained from Hellma, USA. DNA were synthesizedon a Model 392 DNA synthesizer from Perkin-Elmer/ABI. TLC ChromatographyTank (Fisher), and TLC plates (Si250F, JTBaker). A Savant SpeedVac wasused for DNA prep, as are Sep-pak C-18 purification cartridges (Waters),UV lamp, a vortex, 10 cc syringes, and various pipetters (2, 20, 200,1000 μL)

Oligonucleotides were synthesized from natural (A, G, C, and T)nucleotides (Perkin-Elmer/ABI) and isoC, and isoG (EraGen Biosciences,Inc., Madison, Wis.). The synthesized self-complementary andnon-self-complementary sequences are in tables 3 and 4.

TABLE 3 Self-Complementary Sequences (isoC = X ,  isoG = Y) 3AGGA CGT CC Control 3B GGA YXT CC Tandem isoC-isoG effect 3C GXA YXT YCIsoC-isoG in penultimate position 3D GGA GCT CC Control 3E GGA XYT CCswapped tandem isoC-isoG effect

TABLE 4 Non-Self-Complementary Sequences (isoC = X , isoG = Y) 4ASEQ ID NO: 99 5′ GCC AGT TTA A 3′ control 3′ CGG TCA AAT T 5′ 4BSEQ ID NO: 100 5′ GCC AXT TTA A 3′ Single isoC-isoG in AT, TA 3′CGG TYA AAT T 5′ context 4C SEQ ID NO: 101 5′ GCX AGT TTA A 3′Single isoC-isoG in mixed 3′ CGY TCA AAT T 5′ GC and AT context 4DSEQ ID NO: 102 5′ GYC AGT TTA A 3′ Single isoC-isoG in mixed 3′CXG TCA AAT T 5′ GC and CG context 4E SEQ ID NO: 103 5′ GYY AGT TTA A 3′Final tandem isoC-isoG 3′ CXX TCA AAT T 5′ substitution

The following reagents were used in the purification of theoligonucleotides and melting experiments: TLC purification was performedby eluting for 5-6 hrs with n-propanol/ammonia/water (55:35:10 byvolume) (Chou, S.-H., Flynn, P., and Reid, B. (1989) Biochemistry 28,2422-2435, incorporated herein by reference). Hybridization experimentswere carried out in degassed 1×SL Buffer (1.0M NaCl (Fisher), 10 mMsodium cacodylate (Fisher), 0.5 mM Na₂EDTA (Fisher), pH 7) (SantaLucia,J., Allawi, H., and Seneviratne, P. A., (1996) Biochemistry 35,3555-3562, incorporated herein by reference).

Determination of thermodynamic parameters were obtained from meltingcurve data using Meltwin™ v3.0 as described in Petersheim, M., andTurner, D. H. (1983) Biochemistry 22, 253-263, incorporated herein byreference.

After synthesis the oligonucleotides were deprotected in ammonia at 50°C. overnight, lyophilized and purified by TLC by dissolving each samplein 175 μL ddH₂0 and eluting for 5-6 hours. The most intense, leastmobile band was visualized, scraped from the plate, and eluted threetimes with 3 mL ddH₂0. The oligonucleotides were further desalted andpurified with the Sep-pak™ columns by eluting with 30% acetonitrile, 10mM ammonium bicarbonate, pH 7 (SantaLucia, J., Allawi, H., andSeneviratne, P. A., (1996) Biochemistry 35, 3555-3562), and finallydried in the SpeedVac™.

Self-complementary oligonucleotides were quantified and 2.0 OD₂₆₀ ofeach was collected and re-dried in the SpeedVac™. Oligonucleotides werethen diluted in series to provide a one hundred fold dilution series in1×SL Buffer. Absorbance vs. temperature profiles were measured with theBeckman DU-7500 spectrophotometer utilizing the various custommicro-cuvettes, sample carriage and temperature controller. See tables 5and 6 for sample dilution series. The dilution series were prepared foreach of the samples of Tables 3 and 4.

TABLE 5 Series A Place into Sample volume (μL) Add(μL) cuvette(μL) A10.0 94.5 34.5 A2 57.5 40.2 34.5 A3 63.2 44.3 34.5 A4 73.0 51.2 69.0 A555.2 38.5 69.0

After running samples A1-A5 the dilutions for the second series wereassembled. For Series B, the remaining 24.7 μL from the last sample wascombined with the dilutions in cuvettes A-3, A-4 and A-5(˜172.5 μLtotal) and an additional 345 μL of 1×SL Buffer.

TABLE 6 Series B Place into Sample volume (μL) Add(μL) cuvette(μL) B1542.2 0.0 172.5 B2 369.8 230.0 172.5 B3 427.2 270.0 345.0 B4 352.5 224.0345.0 B5 231.5 132.2 345.0

The volumes placed in the cuvettes leaving approximately 4% head spacein each cuvette for thermoexpansion of the samples during the melts.

For each run the samples were further degassed and then annealed byraising the temperature to 85° C. for five minutes, and then cooled to10° C. over five more minutes. To limit condensation, a blanket of dryargon was utilized at low temperatures. For series A and B, measurementswere taken at 260 nm and 280 nm, simultaneously. Samples were heated ata constant rate from 10° C. to 90° C. at 1.0° C./min.

The data collected from the melting experiment were then analyzed withthe Meltwin™ software by curve fit analysis of Tm⁻¹ vs ln(C_(T)), whereC_(T) is the total strand concentration and Tm⁻¹ is the reciprocalmelting temperature (Borer, P. N., Dengler, B., Tinoco, I., Jr., andUhlenbeck, O. C. (1974) J. Mol. Biol. 86, 843-853, incorporated hereinby reference).

Non-self-complementary oligonucleotides were combined in equal molaramounts to 2.0 OD₂₆₀ (optical density at 260 nm) and diluted in the samemanner as the self-complementary oligonucleotides dilution series inTables 5 and 6. Similar melt data was collected and analyzed withMeltwin™ for the non-self-complementary oligonucleotides.

The resulting thermodynamic parameters determined by Meltwin™ for theself-complementary and non-self-complementary oligonucleotides aresummarized in Tables 7 and 8.

TABLE 7 Self-Complementary Sequences Thermodynamic Data(isoC =X , isoG = Y) −ΔG₃₇ −ΔH −ΔS T_(M)(° C.) (kcal/mol) (kcal/mol)(cal/K•mol) 1.0e−4M 1A GGA CGT CC  8.27 53.5 145.9 52.8 1B GGA YXT CC 9.41  57.62 155.4 58.5 1C GXA CGT YC 10.89  66.27 178.6 63.5 1DGGA GCT CC  8.10  51.04 138.5 52.4 lE GGA XYT CC  9.70  57.77 155.0 60.2

TABLE 8 Non-Self-Complementary Sequences Thermodynamic Data (isoC =X, isoG = Y) −ΔG₃₇ −ΔH −ΔS T_(M)(° C.) (kcal/mol) (kcal/mol) (cal/K•mol)1.0e−4M 4A SEQ ID 5′ GCC AGT TTA A 3′  8.43 69.22 196.0 45.8 NO: 99 3′CGG TCA AAT T 5′ 4B SEQ ID 5′ GCC AXT TTA A 3′  9.56 56.66 151.9 54.5NO: 100 3′ CGG TYA AAT T 5′ 4C SEQ ID 5′ GCY AGT TTA A 3′  9.36 62.98172.9 51.6 NO: 101 3′ CGX TCA AAT T 5′ 4D SEQ ID 5′ GYC AGT TTA A 3′ 9.62 54.30 144.1 55.7 NO: 102 3′ CXG TCA AAT T 5′ 4E SEQ ID 5′GYY AGT TTA A 3′ 10.59 70.19 192.2 56.0 NO: 103 3′ CXX TCA AAT T 5′

All samples have concentration dependant T_(M)s and monophasic meltingtransitions. IsoC and isoG contributions to duplex formation appear tobe substantial, adding up to an additional 5° C. (Sample 3B and 4C) perisoC/isoG pair to 10° C. (Sample 3C and 4E) compared to natural (A, G,C, and T) Watson-Crick oligonucleotides.

Tables 7 and 8 show some the extent of the nearest-neighbor effects thatare occurring when AEGIS bases are mixed with natural DNA.

Example 3 and Comparative Example Site Gated Incorporation

First primer (SEQ ID NO: 154) 5′AGAACCCTTTCCTCTTCC Target(SEQ ID NO: 155) 5′AAGAACCCTTTCCTCTTCCGATGCAGGATACTTAACAATAAATATTTSecond Primer (SEQ ID NO: 156)CTACGTCCTATGAATTGTTATTTATAAAXAGGACAGACG 5′X=isoCTP

The sequences of the first primer, target, and second primer are shownin SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156, respectively.

PCR was performed using the following mixture: 0.2 μM first primer, 0.2μM second primer, 50 fM target, 50 μM each dGTP, dATP, dTTP and dCTP, 10mM Tris pH 8, 0.1% BSA, 0.1% Triton X-100, 0.1 μg/μl degraded herringsperm DNA, 40 mM KAc, 2 mM MgCl₂, 1 U Amplitaq Stoffel (Perkin ElmerBiosciences, Foster City, Calif.) in a 20 μl reaction volume. Themixture was held for 2 minutes at 95° C. Then was cycled 30 timesbetween 95° C. with a 1 second hold and 58° C. with a 10 second hold.Finally, the mixture was held for 2 minutes at 58° C.

Two PCR reaction mixtures were prepared. Each PCR reaction mixture wasdesalted using an AutoSeg™ G-50 microspin column (Amersham PharmaciaBiotech Inc., Piscataway, N.J.) to remove unincorporated dNTP's, thecolumn buffer had been exchanged for ddH₂O prior to desalting thesample. The desalted samples were adjusted to these final concentrationsfor the following reaction components: 10 mM Tris pH 8, 0.1% BSA, 0.1%Triton X-100, 0.1 μg/μl degraded herring sperm DNA, 40 mM KAc, 2 mMMgCl₂, 1 U/reaction Amplitaq Stoffel (Perkin Elmer Biosciences, FosterCity, Calif.), and 10 μM Cy3-dTTP (NEN Life Science Products, Inc.,Boston, Mass.) in a 25 μl reaction volume. In addition, disoGTP wasadded in the following concentrations: 0 μM (Comparative Example) or 40μM (Example 3). The reaction mixtures were incubated at 68° C. for 15minutes, and 5 μl of the resulting reactions were examined byelectrophoresis on a 10% denaturing polyacrylamide gel. The gel wasimaged for Cy3 containing extension products using a 595 Fluorimager(Molecular Dynamics, Sunnyvale, Calif.).

The results (data not shown) indicated that there was no additionalextension of the first primer during the final PCR step when disoGTP wasnot present (i.e., there was little or no misincorporation of basesopposite the iso-C of the second primer).

Example 4 Synthesis of Labeled deoxyisoGuanosine 5′-Triphosphates

For the following chemical reactions, tributylammonium pyrophosphate waspurchased from Sigma; biotin N-hydroxysuccinimide ester, was purchasedfrom Pierce Chemical Company; all other chemicals were purchased fromAldrich Chemical Co. or Fisher Chemical Co. and were used withoutfurther purification. Solvents were dried over 4 Å molecular sieves.Reactions were carried out under dry argon in oven-dry glassware. Columnchromatography was performed with silica gel (230-425 mesh).

Abbreviations:

-   Ac₂O Acetic anhydride-   DMF N,N-Dimethylformamide-   DMAP 4,4′-Dimethylaminopyridine-   DMT 4,4′-Dimethoxytrityl-   Et₃N Triethylamine-   MeCN Acetonitrile-   MeOH Methyl alcohol-   Tol p-Toluyl

1-(p,p′-Dimethoxytrityl)-hexamethylenediamine (2)

Hexamethylenediamine (10 eq., 375 mmol, 43.5 g) was coevaporated twotimes from pyridine and dissolved in 100 ml pyridine. DMAP (0.1 eq.,3.75 mmol, 457 mg) was added and the reaction flask placed in an icebath. DMT-chloride (1 eq., 37.5 mmol, 12.69 g), dissolved in 100 mlpyridine, was added dropwise over 2 h. It was stirred at roomtemperature for 4 h, MeOH (5 ml) added, the reaction mixtureconcentrated and the remaining residue extracted with aqueousNaHCO₃/ethyl acetate. The organic layer was washed twice with aqueousNaHCO₃ solution, dried and the solvent evaporated. The obtained productwas used in next step without further purification.

Yield: 14.895 g (35.634 mmol, 95%) sticky oil.

2-Chloro-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-2′-deoxy-3′,5′-ditoluoylriboside(3)

Compound 2 (1.3 equiv., 31.916 mmol, 13.34 g) was coevaporated with DMFand dissolved in 100 ml DMF. Diisopropylethylamine (3.9 equiv., 95.748mmol, 16.65 ml) and compound 1 (1 equiv., 24.551 mmol, 13.282 g),dissolved in 100 ml DMF, were added and it was stirred at roomtemperature for 3 h. It was concentrated, the residue extracted withaqueous NaHCO₃/ethyl acetate, the organic layer dried and the solventevaporated. The residue was triturated with ether twice and the obtainedsolid product used further after drying in vacuum without furtherpurification.

2-Benzyloxy-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-2′-deoxyriboside(4)

Compound 3 (1 equiv., 19.23 mmol, 17.74 g) was dissolved in DMF (25 ml)and added to a solution of NaH (10 eq., 192.3 mmol, 7.69 g of a 60%dispersion in mineral oil) in benzylalcohol (128 mL). The reactionmixture was heated (120° C., 6 h) and then stirred at room temperature(15 h) before filtrated over Celite, the filtrate evaporated, theresidue extracted (ethyl acetate/water), the organic layer washed(NaHCO₃-solution), dried, the solvent evaporated and the residuetriturated 5 times with ether/hexane 1:10. TLC: CHCl₃/10% MeOHR_(F)=0.26.

Yield: 10.280 g (13.562 mmol, 70.5% for 2 steps) foam.

2-Benzyloxy-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-2′-deoxy-5′-O-p,p′-dimethoxytritylriboside(5)

Compound 4 (14.7388 mmol, 11.172 g) was coevaporated with pyridine,dissolved in 150 ml pyridine and DMAP (0.25 equiv., 3.6847 mmol, 450 mg)added. The flask was placed in an ice bath and DMTCI (1.5 equiv., 22.108mmol, 7.484 g) was added slowly over 2 h. It was stirred at roomtemperature for 22 h, then MeOH (1 ml) added, the reaction mixtureconcentrated and the residue extracted (chloroform/aqueous NaHCO₃). Theorganic layer was dried, the solvent evaporated and the residuetriturated with ether/hexane 1:1 to remove the excess DMT and theinsoluble solid product was dried and used further without additionalpurification.

Yield: 14.890 g (14.047 mmol, 95%) light brown foam.

2-Benzyloxy-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-3′-O-acetyl-2′-deoxy-5′-O-p,p′-dimethoxytritylriboside(6)

Compound 5 (14.047 mmol, 14.89 g) was coevaporated with pyridine,dissolved in 200 ml pyridine and DMAP (0.25 equiv., 3.5117 mmol, 428mg), Et₃N (5 equiv., 70.235 mmol, 9.7 ml) and Ac₂O (2.5 equiv., 35.1175mmol, 3.582 g) were added. It was stirred at room temperature for 4.5 h,then MeOH (2 ml) added, the reaction mixture concentrated and theresidue extracted (ethyl acetate/aqueous NaHCO₃). The organic layer wasdried, the solvent evaporated and the residue purified by columnchromatography using an one step gradient of ethyl acetate/hexane/Et₃N30:60:1, then 65:35:3. Yield: 5.93 g (5.385 mmol, 38%), yellow foam.

2-Benzyloxy-6-(6-aminohexyl)-aminopurine-3′-O-acetyl-2′-deoxyriboside(7)

Compound 6 (2.471 mmol, 2.723 g) was dissolved in 50 ml acetonitrile/2ml water and Ce(NH₄)₂(NO₃)₃ (0.3 equiv., 0.74 mmol, 406 mg) was added.It was refluxed for 45 min., then another 0.15 equiv. Ce(NH₄)₂(NO₃)₃(0.37 mmol, 205 mg) added and refluxing continued for 1 h. Then, it wasevaporated, the residue triturated with ether to remove the DMT, theinsoluble product dried and used further without additionalpurification.

2-Benzyloxy-6-(6-trifluoroacetamidohexyl)-aminopurine-3′-O-acetyl-2′-deoxyriboside(8)

The above obtained compound 7 (max. 5.385 mmol) was dissolved in 30 mlMeOH/50 ml ethyl trifluoroacetate/5 ml Et₃N and the reaction mixturestirred at room temperature for 21.5 h. TLC (chloroform/17.5 MeOH):R_(F)=0.72) indicated complete conversion. It was evaporated, theresidue extracted (brine/ethyl acetate), the organic layer dried, thesolvent evaporated and the residue purified by silica gel columnchromatography using a one step gradient of chloroform/1.5% MeOH, then17.5% MeOH. Yield: 2.80 g (4.714 mmol, 87%) foam.

2-Benzyloxy-6-(6-trifluoroacetamidohexyl)-aminopurine-3′-O-acetyl-5′-triphosphoryl-2′-deoxyriboside(9)

Imidazole (61 eq., 306 mg, 4.5 mmol, recrystallised) was dissolved inacetonitrile (3.6 mL) and chilled (0° C.). POCl₃ (19 eq., 0.128 mL) andtriethylamine (61 eq., 0.633 mL) were then added and the mixture wasstirred (0° C., 0.5 h) before adding a portion (0.309 mL) to 8 (1 eq.,0.074 mmol, 44 mg). This mixture was stirred (r.t., 0.5 h) before addingDMF (1.5 mL) containing tributylammonium pyrophosphate (2 eq., 0.16mmol, 73 mg). The reaction was then quenched (2 mL, 10% NH₄COO) 24 hlater and lyophillized. Product was purified by anion-exchangechromatography (Dionex ProPac SAX-10) using 20% MeCN and a gradient of(NH₄)₂CO₃/20% MeCN. Collected product was repetitively lyophilized toremove excess salt. Yield 0.007 mmol (10%), white solid.

6-(6-aminohexyl)-aminopurine-5′-triphosphoryl-2′-deoxyriboside (10)

Compound 9 (0.007 mmol) was dissolved in methanol (2.5 mL) before addingPd/C (10%, 5 mg) and NH₄COO (0.05 mmol, 31 mg). The suspension wasrefluxed (1 h) before filtering off the catalyst and evaporating thesolvent. The residue was then treated with 28% ammonium hydroxide (1.5mL, 3 h, room temp.) before the reaction was dried and the productpurified by anion-exchange chromatography (Dionex ProPac SAX-10) using20% MeCN and a gradient of (NH₄)₂CO₃/20% MeCN. Collected product wasrepetitively lyophilized to remove excess salt. Yield 0.0063 mmol (90%),white solid.

6-(6-biotinylamidohexyl)-aminopurine-5′-triphosphoryl-2′-deoxyriboside(11)

To 10 (0.88 μmol, triethylammonium salt) in H₂O (40 μL) was added sodiumborate (10.5 μL, 1M, pH 8.5) followed by DMF (216 μL) containing biotinN-hydroxysuccinimide ester (2.6 μmmol, 3 eq.). The reaction proceeded (3h, 55° C.) before it was diluted with 20% MeCN and the product purifiedby anion-exchange chromatography (Dionex ProPac SAX-10) using H₂O and agradient of an NH₄HCO₃ solution. Yields approximately 70%.

Example 5 Multiplexed Genotyping of Genomic DNA Using Incorporation of aLabeled Base and Capture on Solid Support Microspheres

The genotypes of nine polymorphic loci were determined following theamplification, query, and capture of targeted nucleic acid sequencesfrom genomic DNA samples. The first step, a multiplex PCR reaction,included a multiplexed set of paired PCR primers. Each pair of PCRprimers included a first primer A and a second primer B that weredesigned to hybridize to and to amplify a region of mouse genomic DNAthat encompasses a known polymorphic site. The second step, a multiplexallele-specific primer extension (ASPE) reaction, included a multiplexedset of tagged allele-specific primers. Each tagged allele-specificprimer included a 5′ tagging sequence containing non-standardnucleotides (iso-G), followed by a c3 (n-propylene) spacer, followed bya 3′ sequence designed to hybridize to one of the DNA strands amplifiedin the previous multiplex PCR step. The allele specificity wasdetermined by the 3′ nucleotide of each tagged allele-specific primer.The multiplexed set of tagged allele-specific primers was designed toquery the set of known polymorphic sites embedded in the set ofmultiplex PCR amplified sequences. A labeled triphosphate (dATP-Biotin)was added to the ASPE reaction, so that allele-specific extension of atagged allele-specific primer led to the incorporation of dATP-Biotin.Unincorporated dATP-Biotin was removed prior to the subsequent capturestep.

The third step, capture of the multiplex ASPE reaction products, usedcapture sequences containing non-standard nucleotides (iso-C) that wereeach covalently coupled to unique Luminex™ microsphere identities. Thecapture sequences were complementary to the tagging sequences used inthe set of tagged allele-specific primers in the preceding ASPEreaction. Phycoerethrin was added to bind to the Biotin label on theextended tagged allele-specific primer strands and provide a fluorescentsignal. Following hybridization between the capture sequences and thetagging sequences, the microspheres were injected into a Luminex100™instrument to detect signal associated with each unique microsphereidentity.

Nine polymorphic regions of the mouse genome were targeted in thisexample:

Target SEQ ID NO: Sequence A/J C57BL6/J  2 104AGAAACAACCATCTAATCCCACACTAAAAT CC TT TCAAGGCTCCACAGACGAAACAGTGAAGATAATTGTTCAGCATACTAACCAACTGATTA CATATTTACCATACTCAGGTTTGTGCTTCATACAAACCCAC/TAGTCCGGCGCTCCCTGTT GATG  3 105CTTCTCCCATTGCCCAGGGCACTCTCCTCT GG AA GTAGAA/GTAGACTGATC/TTTTGTGGAGACATCA  4 106 AGTGCCTGCTACCTGTCAGGTGAAAATTTC CC TTTTAGTGATCCC/TAAGCTCAATGGGTGCYGGGC TTGCAGG  5 107GGTTGGAATGTTTGCACATGCAGTGTTAGT TT CC TATTTGGGC/TGATAACTACTTAGCTTATCTGCCTGGTCCAGC  6 108 CTGATCTGACCTCAGACTGTTGTGCTAACA GG CCGATATAACACCAGTAAGTTGAC/GTCAAAT TGCAGGAAGTAGAGCCTTGC  7 109GACTGCTGGAGAGCTGAGGGAGGCTGTGGA GG AA GAATAAGGAGAGAGCA/GTAGTCTCGTGCCCTGCCCTGCCCATACTGAGCAGCCAAGACAC  8 110 GGACTGTCCAAAKGGATCTCAAGGAGAAT AA GGGTCCTTGCTATTAA/GGAGTATAAAGGCATAA AAGAGGTCATAGGGGACAACCATGACCAAG AAGTTG 9 111 CCTTCCTGCAYTCCACAGTATAAACACAGA AA GGATGCACACTGCA/GGTCGTTGTATTTGTGTTC GATGTGAATTAAAGATGCTTTGGCTAAGCCAGGAGATGATAATACTG 10 112 CACATACACCATGTCAGCCATCAGCGCAAA CC TTGCCTTCGAGTTTCAGCTGTGAGATGAAGGC TTGGAGAAGCACGTTGATCTGCAAAGAAGAAAGGAGCTAGCGGAGGCC/TGGTCACTGACC GACTGCTCA

The following nucleic acids were used in the multiplex PCR step for thisexample:

Nucleic acid SEQ ID component Sequence NO PCR Primer 1A5′-CATCTAACAGGGAGCGCC-3′ 113 PCR Primer 1B5′-6FAM-AGAAACAACCATCTAATCCCACA-3′ 114 PCR Primer 2A5′-6FAM-CTTCTCCCATTGCCCAGG-3′ 115 PCR Primer 2B5′-TGATGTCTCCACAAAGATCAGTC-3′ 116 PCR Primer 3A5′-AGTGCCTGCTACCTGTCAG-3′ 117 PCR Primer 3B 5′-6FAM-CCTGCAAGCCAGCACC-3′118 PCR Primer 4A 5′-6FAM-GGTTGGAATGTTTGCACATGC-3′ 119 PCR Primer 4B5′-GCTGGACCAGGCTAGATAAGC-3′ 120 PCR Primer 5A5′-6FAM-CTGATCTGACCTCAGACTGTTG-3′ 121 PCR Primer 5B5′-GCAAGGCTCTACTTCCTGC-3′ 122 PCR Primer 6A5′-6FAM-GACTGCTGGAGAGCTGAGG-3′ 123 PCR Primer 6B5′-GTGTCTTGGCTGCTCAGTATG-3′ 124 PCR Primer 7A5′-6FAM-GGACTGTCCAAAGGGATCTC-3′ 125 PCR Primer 7B5′-CAACTTCTTGGTCATGGTTGTC-3′ 126 PCR Primer 8A5′-Cy3-CCTTCCTGCAYTCCACAG-5 ′ 127 PCR Primer 8B5′-6FAM-CAGTATTATCATCTCCTGGCTTAGC-3′ 128 PCR Primer 9A5′-6FAM-CACATACACCATGTCAGCC-3′ 129 PCR Primer 9B5′-TGAGCAGTCGGTCAGTG-3 ′ 130 Template 1 Mouse genomic DNA; Strain: A/JTemplate 2 Mouse genomic DNA; Strain: C57BL6/J

PCR primers were synthesized and diluted in 1 mM MOPS pH 7.5, 0.1 mMEDTA. The 6FAM or Cy3 fluor on some of the PCR primers is added to allowinvestigation of the multiplex PCR reaction on a polyacrylamide gel.

Mouse genomic DNA samples were purchased from the Jackson Laboratory(Bar Harbor, Me.). All genomic DNA samples were diluted to 5 ng/μL in 1mM MOPS pH 7.5, 0.1 mM EDTA. PCR reaction components were:

Component 1X Concentration Supplier and Location 10X PCR Buffer II 1.2 XApplied Biosystems, Foster City, CA MgCl₂ 2 mM Sigma, St. Louis, MO dATP200 μM Amersham dGTP 200 μM Amersham dCTP 200 μM Amersham dTTP 200 μMAmersham Amplitaq ™ Gold 0.1 U/μL Applied Biosystems, DNA PolymeraseFoster City, CA PCR Primers (each) 0.1 μM

A Master Mix of all listed components was prepared at 1.09×concentration for 25 μL final reaction volumes. 23 μL Master Mix wascombined with 2 μL of genomic DNA template (5 ng/μL) in individual PCRtubes. A negative control included water in place of genomic DNAtemplate. PCR reactions were cycled as follows:

Cycle # Step Temp Time 1 1 95° C. 9 minutes 2-41 1 95° C. 5 seconds 255° C. 30 seconds 3 62° C. 30 seconds 42 1 62° C. 5 minutes 43 1  4° C.hold

Following PCR cycling, 2 μL of each PCR reaction was transferred to actas template in the multiplex ASPE reaction. The following syntheticnucleic acids were used as tagged allele-specific (TAS) primers in themultiplex ASPE reaction:

Nucleic acid SEQ ID component Sequence NO TAS Primer 15′-GTGYACAYGC-c3-GCTTCATACAAACCCAC-3′ 131 TAS Primer 25′-CGAYTCTGYC-c3-GCTTCATACAAACCCAT-3′ 132 TAS Primer 35′-CTAYCAAYCC-c3-CACTCTCCTCTGTAGAA-3′ 133 TAS Primer 45′-GAGAYCYAAG-c3-CACTCTCCTCTGTAGAG-3′ 134 TAS Primer 55′-GTTCYTGAYG-c3-GAAAATTTCTTAGTGATCCT-3′ 135 TAS Primer 65′-GCYTAYCTAC-c3-AAAATTTCTTAGTGATCCC-3′ 136 TAS Primer 75′-GTTAYCYTCC-c3-AGTGTTAGTTATTTGGGT-3′ 137 TAS Primer 85′-CACYATACYG-c3-GTGTTAGTTATTTGGGC-3′ 138 TAS Primer 95′-CYTACCYATG-c3-TAACACCAGTAAGTTGAC-3′ 139 TAS Primer 105′-GYCGAYAATC-c3-TAACACCAGTAAGTTGAG-3′ 140 TAS Primer 115′-GYCGTAYTTG-c3-AGAATAAGGAGAGAGCA-3 ′ 141 TAS Primer 125′-GTYTATYCCG-c3-GAATAAGGAGAGAGCG-3′ 142 TAS Primer 135′-GACAYACYTC-C3-AGAATAGTCCTTGCTATTAA-3′ 143 TAS Primer 145′-GGAAYAACYG-C3-AGAATAGTCCTTGCTATTAG-3′ 144 TAS Primer 155′-GATYTYCAGC-c3-AGAATGCACACTGCA-3 ′ 145 TAS Primer 165′-GTYATYTGCG-c3-GAATGCACACTGCG-3′ 146 TAS Primer 175′-GATYGTCYYG-c3-GCTAGCGGAGGCC-3′ 147 TAS Primer 185′-GGYCTYATGG-c3-GCTAGCGGAGGCT-3′ 148

Components of the ASPE reaction were:

Component 1X Concentration Supplier and Location Bis-Tris-Propane pH 10mM Sigma, St. Louis, MO 8.9 Potassium Acetate 40 mM Sigma, St. Louis, MOMgCl₂ 2 mM Sigma, St. Louis, MO Biotin-11-dATP, 4 μM NEN, Boston, MAdGTP 200 μM Amersham Pharmacia, Piscataway, NJ dCTP 200 μM AmershamPharmacia, Piscataway, NJ dTTP 200 μM Amersham Pharmacia, Piscataway, NJAmplitaq ™ Gold 0.067 U/μL Applied Biosystems, DNA Polymerase FosterCity, CA TAS Primers (each) 0.067 μM EraGen Biosciences, Inc., Madison,WI

A Master Mix containing all of the above components except TAS primerswas prepared at 1.36×. Each ASPE reaction consisted of 11 μL Master Mix,2 μL multiplex TAS primer mix (0.5 μM each), 2 μL PCR reaction (fromprevious step). ASPE reactions were cycled as follows:

Cycle # Step Temp Time 1 1 95° C. 12 minutes 2-5 1 95° C.  3 seconds 248° C. 15 seconds slow 30 degrees ramp per minute 3 62° C. 30 seconds 61  4° C. hold

Following ASPE cycling, 10 μL of the reaction volume was combined with 5μL of a solution containing 40 mM Tris, 40 mM EDTA to stop the activityof the polymerase. The reaction was purified over a G-50 column toremove unincorporated dATP-Biotin. The purified multiplex ASPE reactionwas then deconvoluted by capture sequences coupled to Luminex™microspheres (Luminex Corp, Houston, Tex.). The coupled microsphereswere:

Microsphere SEQ Capture Identity ID NO: Sequence  1  2 CXGTTXTTCC  2  9CATXGGTAXG  7 14 CGGXATAXAC 15 13 CAAXTACGXC 17 22 GCTGXAXATC 18 23CGCAXATXAC 19  1 GAXGTXTGTC 20  3 GGXTTGXTAG 21  4 CTTXGXTCTC 22  5CXTCAXGAAC 34  7 GGAXGXTAAC 35  8 CXGTATXGTG 37  6 GTAGXTAXGC 38 10GATTXTCGXC 45 28 CXXGACXATC 47 29 CCATXAGXCC 61 36 GCXTGTXCAC 62 37GXCAGAXTCG

The coupled microspheres were combined in a mixture containing equalnumbers of each microsphere identity in a storage buffer (10 mM MOPS pH7.5, 200 mM NaCl, 1 mM EDTA, 1% PEG8000, 0.05% SDS). The components ofthe capture hybridization reaction were:

Component 1X Concentration Supplier and Location MOPS pH 7.5 10 mMFisher Chemical, Fair Lawn, NJ NaCl 200 mM Fisher Chemical, Fair Lawn,NJ MgCl₂ 50 mM Sigma, St. Louis, MO EDTA 1 mM Fisher Chemical, FairLawn, NJ PEG8000    1% Sigma, St. Louis, MO SDS 0.05% Fisher Chemical,Fair Lawn, NJ Herring sperm DNA 0.1 mg/mL Promega, Madison, WIMicrosphere mix 1000 each EraGen Biosciences, Inc., identity Madison, WI

A Master Mix of all listed components was prepared at 1.2× concentrationfor 60 μL final reaction volume. 50 μL Master Mix was combined with 10μL of the purified multiplex ASPE reaction and allowed to hybridize atroom temperature for 10 minutes. 10 μL of a 0.01 mg/mL solution ofStreptavidin Phycoerethrin (Molecular Probes, Eugene, Oreg.) inhybridization buffer (10 mM MOPS pH7.5, 200 mM NaCl, 50 mM MgCl₂, 1 mMEDTA, 1% PEG8000, 0.05% SDS) was added to each capture hybridizationreaction prior to injection into a Luminex100™ instrument.

For each capture hybridization reaction, 55 μL was injected into theLuminex100™ at a rate of 60 μL/min and the read continued until 50 ofeach microsphere identity were counted. The median fluorescenceintensity was used as a measurement of the fluorescent signal associatedwith each microsphere identity. The results are shown in FIG. 14.

Example 6 Multiplexed Genotyping of Genomic DNA Using Site-specificIncorporation of a Labeled Non-standard Base and Capture on SolidSupport Microspheres

The genotypes of nine polymorphic loci were determined following theamplification, query, and capture of targeted nucleic acid sequencesfrom genomic DNA samples. The first step, a multiplex PCR reaction,included a multiplexed set of paired PCR primers. Each pair of PCRprimers included a first primer A and a second primer B, and the secondprimer B included a non-standard nucleotide (iso-C) near its 5′ end.Each primer pair was designed to hybridize to and to amplify a region ofmouse genomic DNA that encompasses a known polymorphic site. The nextstep, a multiplex allele-specific primer extension (ASPE) reaction,included a multiplexed set of tagged allele-specific primers. Eachtagged allele-specific primer included a 5′ tagging sequence containingnon-standard nucleotides (iso-G), followed by a c3 (n-propylene) spacer,followed by a 3′ sequence designed to hybridize to one of the DNAstrands amplified in the previous multiplex PCR step. The allelespecificity was determined by the 3′ nucleotide of each taggedallele-specific primer. The multiplexed set of tagged allele-specificprimers was designed to query the set of known polymorphic sitesembedded in the set of multiplex PCR amplified sequences. A labelednon-standard triphosphate (isoGTP-Biotin) was added to the ASPEreaction, so that allele-specific extension of a tagged allele-specificprimer led to the incorporation of the labeled non-standard triphosphate(isoGTP-Biotin) opposite the non-standard nucleotide (iso-C) in thetemplate strand created in the preceding multiplex PCR reaction.Unincorporated isoGTP-Biotin was removed prior to the subsequent capturestep.

The third step, capture of the multiplex ASPE reaction products, usedcapture sequences containing non-standard nucleotides (iso-C) that wereeach covalently coupled to unique Luminex™ microsphere identities. Thecapture sequences were complementary to the tagging sequences used inthe set of tagged allele-specific primers in the preceding ASPEreaction. Phycoerethrin was added to bind to the Biotin label on theextended tagged allele-specific primer strands and provide fluorescentsignal. Following hybridization between the capture sequences and thetagging sequences, the microspheres were injected into a Luminex100™instrument to detect signal associated with each unique microsphereidentity.

Nine polymorphic regions of the mouse genome were targeted in thisexample:

SEQ Target ID NO: Sequence A/J C57BL6/J AB6F1  2 104AGAAACAACCATCTAATCCCACACTAAAAT CC TT CT TCAAGGCTCCACAGACGAAACAGTGAAGAATAATTGTTCAGCATACTAACCAACTGATTA CATATTTACCATACTCAGGTTTGTGCTTCATACAAACCCAC/TAGTCCGGCGCTCCCTGTTA GATG  3 105CTTCTCCCATTGCCCAGGGCACTCTCCTCT GG AA AG GTAGAA/GTAGACTGATYTTTGTGGAGACATCA  4 106 AGTGCCTGCTACCTGTCAGGTGAAAATTTC CC TT CTTTAGTGATCCC/TAAGCTCAATGGGTGCYGGC TTGCAGG  5 107GGTTGGAATGTTTGCACATGCAGTGTTAG TT CC CT TATTTGGGC/TGATAACTACTTAGCTTATCGCCTGGTCCAGC  6 108 CTGATCTGACCTCAGACTGTTGTGCTAACA GG CC CGGATATAACACCAGTAAGTTGAC/GTCAAATAC TGCAGGAAGTAGAGCCTTGC  7 109GACTGCTGGAGAGCTGAGGGAGGCTGTGGA GG AA AG GAATAAGGAGAGAGCA/GTAGTCTCGTGCCCTGCCCTGCCCATACTGAGCAGCCAAGACAC GGACTGTCCAAAKGGATCTCAAGGAGAATA  8 110GTCCTTGCTATTAA/GGAGTATAAAGGCATAA AA GG AG AAGAGGTCATAGGGGACAACCATGACCAAGAAGTTG  9 111 CCTTCCTGCAYTCCACAGTATAAACACAG AA GG AGATGCACACTGCA/GGTCGTTGTATTTGTGT GATGTGAATTAAAGATGCTTTGGCTAAGCAGGAGATGATAATACTG 10 112 CACATACACCATGTCAGCCATCAGCGCAA CC TT CTGCCTTCGAGTTTCAGCTGTGAGATGAAGG TTGGAGAAGCACGTTGATCTGCAAAGAAGCAAAGGAGCTAGCGGAGGCC/TGGTCACTGACC GACTGCTCA

The following nucleic acids were used in the multiplex PCR step for thisexample:

Nucleic acid SEQ ID component Sequence NO PCR Primer 1A5′-6FAM-AGAAACAACCATCTAATCCCACA-3′ 114 PCR Primer 1B5′-TXCATCTAACAGGGAGCGCC-3 ′ 157 PCR Primer 2A5′-6FAM-CTTCTCCCATTGCCCAGG-3′ 115 PCR Primer 2B5′-TXTGATGTCTCCACAAAGATCAGTC-3′ 158 PCR Primer 3A5′-6FAM-CCTGCAAGCCAGCACC-3′ 118 PCR Primer 3B 5′-TXCCTGCAAGCCAGCACC-3′159 PCR Primer 4A 5′-6FAM-GGTTGGAATGTTTGCACATGC-3′ 119 PCR Primer 4B5′-TXGCTGGACCAGGCTAGATAAGC-3′ 160 PCR Primer 5A5′-6FAM-CTGATCTGACCTCAGACTGTTG-3′ 121 PCR Primer 5B5′-TXGCAAGGCTCTACTTCCTGC-3′ 161 PCR Primer 6A5′-6FAM-GACTGCTGGAGAGCTGAGG-3′ 123 PCR Primer 6B5′-TXGTGTCTTGGCTGCTCAGTATG-3′ 162 PCR Primer 7A5′-6FAM-GGACTGTCCAAAGGGATCTC-3′ 125 PCR Primer 7B5′-TXCAACTTCTTGGTCATGGTTGTC-3 ′ 163 PCR Primer 8A5′-6FAM-CAGTATTATCATCTCCTGGCTTAGC-3′ 128 PCR Primer 8B5′-TXCCTTCCTGCACTCCACAG-3′ 164 PCR Primer 9A5′-6FAM-CACATACACCATGTCAGCC-3′ 129 PCR Primer 9B5′-TXTGAGCAGTCGGTCAGTG-3′ 165 Template 1 Mouse genomic DNA; Strain: A/JTemplate 2 Mouse genomic DNA; Strain: C57BL6/J Template 3Mouse genomic DNA; Strain: AB6F1

PCR primers were synthesized and diluted in 1 mM MOPS pH 7.5, 0.1 mMEDTA. Mouse genomic DNA samples were purchased from the JacksonLaboratory in (Bar Harbor, Me.). All genomic DNA samples were diluted to5 ng/μL in 1 mM MOPS pH 7.5, 0.1 mM EDTA. PCR reaction components were:

Component 1X Concentration Supplier and Location 10X PCR Buffer II 1.2 XApplied Biosystems, Foster City, CA MgCl₂ 2 mM Sigma, St. Louis, MO DATP200 μM Amersham DGTP 200 μM Amersham DCTP 200 μM Amersham DTTP 200 μMAmersham Amplitaq ™ Gold 0.1 U/μL Applied Biosystems, DNA PolymeraseFoster City, CA PCR Primers (each) 0.2 μM

A Master Mix of all listed components was prepared at 1.09×concentration for 25 μL final reaction volumes. 23 μL Master Mix wascombined with 2 μL of genomic DNA template (5 ng/μL) in individual PCRtubes. A negative control included water in place of genomic DNAtemplate. PCR reactions were cycled as follows:

Cycle # Step Temp Time  1 1 95° C.  9 minutes 2-41 1 95° C. 10 seconds 255° C. 10 seconds 3 70° C. 30 seconds 42 1 70° C.  5 minutes 43 1  4° C.hold

Following PCR cycling, 2 μl, of each PCR reaction was transferred to actas template in the multiplex ASPE reaction. The following syntheticnucleic acids were used as tagged allele-specific (TAS) primers in themultiplex ASPE reaction:

Nucleic acid SEQ ID component Sequence NO TAS Primer 15′-GTGYACAYGC-c3-GCTTCATACAAACCCAC-3′ 131 TAS Primer 25′-CGAYTCTGYC-c3-GCTTCATACAAACCCAT-3′ 132 TAS Primer 35′-CTAYCAAYCC-c3-CACTCTCCTCTGTAGAA-3′ 133 TAS Primer 45′-GAGAYCYAAG-c3-CACTCTCCTCTGTAGAG-3′ 134 TAS Primer 55′-GTTCYTGAYG-c3-GAAAATTTCTTAGTGATCCT-3′ 135 TAS Primer 65′-GCYTAYCTAC-c3-AAAATTTCTTAGTGATCCC-3′ 136 TAS Primer 75′-GTTAYCYTCC-c3-AGTGTTAGTTATTTGGGT-3′ 137 TAS Primer 85′-CACYATACYG-c3-GTGTTAGTTATTTGGGC-3′ 138 TAS Primer 95′-CYTACCYATG-c3-TAACACCAGTAAGTTGAC-3′ 139 TAS Primer 105′-GYCGAYAATC-c3-TAACACCAGTAAGTTGAG-3′ 140 TAS Primer 115′-GYCGTAYTTG-c3-AGAATAAGGAGAGAGCA-3′ 141 TAS Primer 125′-GTYTATYCCG-c3-GAATAAGGAGAGAGCG-3′ 142 TAS Primer 135′-GACAYACYTC-C3-AGAATAGTCCTTGCTATTAA-3′ 143 TAS Primer 145′-GGAAYAACYG-C3-AGAATAGTCMGCTATTAG-3′ 144 TAS Primer 155′-GATYTYCAGC-c3-AGAATGCACACTGCA-3′ 145 TAS Primer 165′-GTYATYTGCG-c3-GAATGCACACTGCG-3′ 146 TAS Primer 175′-GATYGTCYYG-c3-GCTAGCGGAGGCC-3′ 147 TAS Primer 185′-GGYCTYATGG-c3-GCTAGCGGAGGCT-3′ 148

Components of the ASPE reaction were:

Component 1X Concentration Supplier and Location Bis-Tris-Propane 10 mMSigma, St. Louis, MO pH 8.9 Potassium Acetate 40 mM Sigma, St. Louis, MOMgCl₂ 2 mM Sigma, St. Louis, MO dATP 50 μM Amersham Pharmacia,Piscataway, NJ dGTP 50 μM Amersham Pharmacia, Piscataway, NJ dCTP 50 μMAmersham Pharmacia, Piscataway, NJ dTTP 50 μM Amersham Pharmacia,Piscataway, NJ d-isoGTP-Biotin 10 μM EraGen Biosciences, Inc., Madison,WI Klentaq DNA 0.067 U/μL Ab Peptides, St. Louis, MO Polymerase TASPrimers (each) 0.067 μM EraGen Biosciences, Inc., Madison, WI

A Master Mix containing all of the above components was prepared at1.15×. Each ASPE reaction consisted of 13 μL Master Mix and 2 μL PCRreaction (from previous step). ASPE reactions were cycled as follows:

Cycle # Step Temp Time  1 1 95° C. 2 minutes 2-11 1 95° C. 1 seconds 248° C. 1 seconds 3 72° C. 1 minutes 12 1 72° C. 5 minutes 13 1  4° C.hold

Following ASPE cycling, 5 μL of a solution containing 40 mM Tris, 40 mMEDTA was added to the multiplex ASPE reaction to stop the activity ofthe polymerase. The reaction was purified over a G-50 column to removeunincorporated d-isoGTP-Biotin. The purified multiplex ASPE reaction wasthen deconvoluted by capture sequences coupled to Luminex™ microspheres(Luminex Corp, Houston, Tex.). The coupled microspheres were:

Microsphere SEQ Capture Identity ID NO: Sequence  1  2 CXGTTXTTCC  2  9CATXGGTAXG  7 14 CGGXATAXAC 15 13 CAAXTACGXC 17 22 GCTGXAXATC 18 23CGCAXATXAC 19  1 GAXGTXTGTC 20  3 GGXTTGXTAG 21  4 CTTXGXTCTC 22  5CXTCAXGAAC 34  7 GGAXGXTAAC 35  8 CXGTATXGTG 37  6 GTAGXTAXGC 38 10GATTXTCGXC 45 28 CXXGACXATC 47 29 CCATXAGXCC 61 36 GCXTGTXCAC 62 37GXCAGAXTCG

The coupled microspheres were combined in a mixture containing equalnumbers of each microsphere identity in a storage buffer (10 mM MOPS pH7.5, 200 mM NaCl, 1 mM EDTA, 1% PEG8000, 0.05% SDS). The components ofthe capture hybridization reaction were:

Component 1X Concentration Supplier and Location MOPS pH 7.5 10 mMFisher Chemical, Fair Lawn, NJ NaCl 200 mM Fisher Chemical, Fair Lawn,NJ MgCl₂ 50 mM Sigma, St. Louis, MO EDTA 1 mM Fisher Chemical, FairLawn, NJ PEG8000   1% Sigma, St. Louis, MO SDS 0.5% Fisher Chemical,Fair Lawn, NJ Herring sperm DNA 0.1 mg/mL Promega, Madison, WIMicrosphere mix 1000 each EraGen Biosciences, Inc., identy Madison, WI

A Master Mix of all listed components was prepared at 1.2× concentrationfor 60 μL final reaction volume. 50 μL Master Mix was combined with 10μL of the purified multiplex ASPE reaction and allowed to hybridize atroom temperature for 10 minutes. 10 μL of a 0.01 mg/mL solution ofStreptavidin Phycoerethrin (Molecular Probes, Eugene, Oreg.) inhybridization buffer (10 mM MOPS pH7.5, 200 mM NaCl, 50 mM MgCl₂, 1 mMEDTA, 1% PEG8000, 0.05% SDS) was added to each capture hybridizationreaction prior to injection into a Luminex100™ instrument.

For each capture hybridization reaction, 55 μL was injected into theLuminex100™ at a rate of 60 μL/min and the read continued until 50 ofeach microsphere identity were counted. The median fluorescenceintensity was used as a measurement of the fluorescent signal associatedwith each microsphere identity. The results are shown in FIG. 15.

The present invention should not be considered limited to the particularexamples described above, but rather should be understood to cover allaspects of the invention as fairly set out in the attached claims.Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable will bereadily apparent to those of skill in the art to which the presentinvention is directed upon review of the instant specification.

Example 7 Genotyping of Genomic DNA Using Site Specific Ligation of aReporter Oligonucleotide to an Allele Specific Extension Product andCapture on Solid Support Microspheres

The genotype of a polymorphic loci was determined following theamplification, query, and capture of target nucleic acid sequences fromgenomic DNA samples. The first step, a PCR reaction, included a set ofPCR primers: a first primer A and a second primer B. The primer Bcontained a 5′ sequence non-complementary to the target with an iso-C atthe junction of the analyte specific and non-complementary portion. Theprimer pair was designed to hybridize to and amplify a region of mousegenomic DNA that encompasses a known polymorphic site. The second step,an allele specific primer extension (ASPE) reaction, included a set oftagged allele-specific primers. Each tagged allele-specific primer wascomposed of a 5′ tagging sequence containing non-standard nucleotides(iso-G), followed by a c3 spacer, followed by a 3′ sequence designed tohybridize to one of the DNA strands amplified in the previous PCR step.The allele specificity was determined by the 3′ nucleotide of eachtagged allele-specific primer. The set of tagged allele-specific primerswas designed to query a known polymorphic site embedded in the amplifiedsequence. A DNA ligase and a reporter oligonucleotide containing a 5′phosphate, and a 3′ biotin modifications were included in the ASPEreaction. This reporter oligonucleotide was complimentary to the 5′region of primer B used to generate the amplicon that was queried. Thestrand of the amplified product containing this non-standard basecontaining region served as the template for the ASPE reaction. Duringallele specific primer extension, the DNA polymerase terminates at thebase prior to the iso-C in the template strand, thus leaving a singlestranded region to which the reporter oligonucleotide to hybridize. Thecomplex between the extended ASPE primer, the template, and the reporteroligonucleotide results in a nick structure suitable for ligation by aDNA ligase.

The third step, capture of the multiplex ASPE reaction products, usedsequences containing non-standard nucleotides (iso-C) that were eachcovalently coupled to unique Luminex microsphere identities. The capturesequences were complementary to the tagging sequences used in the set oftagged allele-specific primers in the preceding ASPE reaction.Streptavidin-phycoerthrin was added to bind to the biotin label on theextended and ligated allele-specific primer strands to providefluorescent signal. Following hybridization between the capturesequences and the tagging sequences, the microspheres were injected intoa Luminex 100 instrument to detect signal associated with each uniquemicrosphere identity.

A single polymorphic region of the mouse genome was targeted in thisexample:

SEQ ID NO: Target Sequence A/J C57BL6/J AB6F1 1495′CTTCTCCCATTGCCCAGGGCACTCT GG AA AG CCTCTGTAGARTAGACTGATMTGTGGAGACATCA 3′ Nucleic Acid Component Sequence 5′-3′ SEQ ID NO:PCR Primer A PO₄-CTTCTCCCATTGCCCAGG 115 PCR Primer BCXGCXAGXGATXTGATGTCTCCACAAAGATCAGTC 150 Template 1Mouse genomic DNA; Strain: A/J Template 1Mouse genomic DNA; Strain: C57BL6/J Template 1Mouse genomic DNA; Strain: AB6F1

Mouse Genomic DNA samples were purchased from Jackson Laboratories (BarHarbor, Me.). All genomic DNA samples were diluted to 10 ng/ul in 1 mMMOPS pH 7.5, 0.1 mM EDTA. PCR reaction components were:

Component 1X Concentration Supplier and Location 10X PCR 1.2 X AppliedBiosystems, Buffer II Foster City, CA MgCl₂ 2 mM Sigma, St. Louis, MOdGTP 200 uM Promega, Madison, WI dATP 200 uM Promega, Madison, WI dTTP200 uM Promega, Madison, WI dCTP 200 uM Promega, Madison, WI AmplitaqDNA 0.2 U/ul Applied Biosystems, Polymerase Stoffel Foster City, CAFragment PCR Primer A 0.2 uM PCR Primer B 0.2 uM

A master mix of all listed components was prepared at 1.07×concentration for 30 ul final reaction volume. 23 ul of master mix wascombined with 2 ul of genomic DNA template (5 ng/ul) in individual PCRtubes. A negative control included water in place of genomic DNAtemplate. PCR reactions were thermal cycled as follows:

Cycle # Step Temp Time  1 1 95° C.  2 minutes 2-41 1 95° C. 10 seconds 255° C. 10 seconds 3 65° C. 30 seconds 42 1 65° C.  5 minutes 43 1  4° C.hold

Following PCR cycling, 3 ul of 5 U/ul lambda exonuclease (New EnglandBiolabs, Beverly, Mass.) was added to each reaction to remove thenon-template strand of the amplicon created by PCR primer A. Followingaddition of lambda exonuclease the reaction tubes were heated to 37° C.for 5 minutes, then to 95° C. for 2 minutes. Following this digest, 1 ulof each PCR reaction was transferred to act as template in the ASPEreaction. The following nucleic acid sequences were used as taggedallele-specific (TAS) primers in the ASPE reaction:

Nucleic acid SEQ ID component Sequence 5′-3′ NO: TAS Primer 1CTAYCAAYCC-c3-CACTCTCCT 151 CTGTAGAA TAS Primer 2GAGAYCYAAG-c3-CACTCTCCT 152 CTGTAGAG Reporter PO₄-YATCYCTYGCYG-Biotin153 oligonucleotide

Components of the ASPE reaction were:

Component 1X Concentration Supplier and Location 10X PCR 1.2 X AppliedBiosystems, Buffer II Foster City, CA MgCl₂ 2 mM Sigma, St. Louis, MODGTP 200 uM Promega, Madison, WI DATP 200 uM Promega, Madison, WI DTTP200 uM Promega, Madison, WI DCTP 200 uM Promega, Madison, WI AmplitaqDNA 0.1 U/ul Applied Biosystems, Polymerase Foster City, CA StoffelFragment TAS Primer 1 0.1 uM TAS Primer 2 0.1 uM Reporter 0.2 uMoligonucleotide DTT 5 mM Fisher Scientific, Pittsburgh, PA NAD 1 mMRoche, Indianapolis, IN Taq DNA ligase 2 U/ul New England Biolabs,Beverly, MA

A master mix containing all of the above components was prepared at1.11×. Each ASPE reaction consisted of 9 ul master mix and 1 ul PCRreaction (from previous step). ASPE reactions were cycled as follows:

Cycle # Step Temp Time  1 1 95° C. 30 seconds  2-13 1 95° C. 1 seconds 248° C. 1 seconds 3 58° C. 2 minutes 14 1  4° C. hold

Following ASPE cycling the reactions were deconvoluted by capturesequences coupled to Luminex microspheres (Luminex Corp, Austin, Tex.).The coupled microspheres were:

Microsphere SEQ Capture sequence Identity ID NO: 5′-3′ 20 3 GGXTTGXTAG21 4 CTTXGXTCTC

The coupled microspheres were combined in a mixture containing equalnumbers of each microsphere identity in a storage buffer (10 mM MOPS pH7.5, 200 mM NaCl, 1 mM EDTA, 1% PEG8000, 0.05% SDS), The components ofthe capture hybridization reaction were:

Component 1X Concentration Supplier and Location MOPS pH 7.5 10 mMSigma, St. Louis, MO NaCl 200 mM Sigma, St. Louis, MO MgCl₂ 50 mM Sigma,St. Louis, MO EDTA 1 mM Sigma, St. Louis, MO PEG 8000    1% Sigma, St.Louis, MO SDS 0.05% Sigma, St. Louis, MO Herring Sperm DNA 0.1 mg/mlPromega, Madison, WI Microsphere mix 1000 each Luminex Corp., Austin, TXidentity

A master mix containing all of the above components was prepared at 1.2×concentration for a 60 ul final reaction volume. 50 ul of this mastermix was added to each ASPE reaction and allowed to hybridize for 10minutes at room temperature. 10 ul of solution ofstreptavidin-phycoerythrin (0.075 mg/ml in 10 mM MOPS pH 7.5, NaCl 200mM, MgCl₂ 50 mM, EDTA 1 mM, PEG 8000 1%, SDS 0.05) (Molecular Probes,Eugene, Oreg.) was added to each capture hybridization prior toinjection into a Luminex 100 instrument.

For each capture hybridization reaction, 45 ul of the reaction mixturewas injected into the Luminex 100 at a rate of 60 μl/min and the readcontinued until 100 of each microsphere identity were counted. Themedian fluorescence intensity (MFI) was used as a measurement of thefluorescent signal associated with each identity. The results are shownin FIG. 17.

In the above example, the non-standard base of primer B at the junctionof the 5′ non-complementary sequence and the analyte-specific sequenceis designed to prevent extension by the polymerase at the junction ofthe tagging and analyte specific sequences. It is specificallyenvisioned that other suitable linkers may be used to stop thepolymerase, including, for example, 2′-O-methyl bases such as2′-O-methyl ribonucleotides.

What is claimed is:
 1. A method for detecting a target nucleic acid in asample comprising: a) synthesizing a nucleic acid comprising a copy ofthe target nucleic acid or a complement of the target nucleic acid usinga reaction mixture that comprises (i) a first primer comprising atagging sequence and a sequence complementary to a first sequence of thetarget nucleic acid or the complement of the target nucleic acid, (ii) asecond primer comprising at least one non-natural base and a sequencecomplementary to a second sequence of the target nucleic acid or thecomplement of the target nucleic acid, and (iii) a labeled, non-naturalnucleotide triphosphate having a non-natural base that is complementaryto the non-natural base of the second primer; b) contacting thesynthesized nucleic acid with an oligonucleotide bound to a solidsupport, wherein the oligonucleotide comprises a molecular recognitionsequence that is complementary to the tagging sequence of the firstprimer; and c) detecting specific hybridization of the synthesizednucleic acid and the oligonucleotide bound to the solid support, therebydetecting the target nucleic acid.
 2. The method of claim 1, wherein thelabel comprises a coupling agent.
 3. The method of claim 2, wherein thecoupling agent comprises biotin.
 4. The method of claim 3, wherein themethod further comprises contacting the synthesized nucleic acid with astreptavidin-phycoerythrin.
 5. The method of claim 1, wherein the labelcomprises a fluorophore.
 6. The method of claim 1, wherein thenon-natural base is selected from the group consisting of iso-cytosineand iso-guanine.
 7. The method of claim 1, wherein the target nucleicacid is detected quantitatively.
 8. The method of claim 1, wherein thespecific hybridization is detected without performing washing.
 9. Themethod of claim 1, wherein the specific hybridization is detected atroom temperature.
 10. The method of claim 1, wherein the first primercomprises a n-propylene spacer between the tagging sequence and thesequence complementary to a first sequence of the target nucleic acid orthe complement of the target nucleic acid.
 11. The method of claim 1,wherein the tagging sequence and the molecular recognition sequence eachcomprise at least one non-natural base, and further wherein the at leastone non-natural base of the tagging sequence is complementary to the atleast one non-natural base of the molecular recognition sequence. 12.The method of claim 11, wherein the non-natural base is selected fromthe group consisting of iso-cytosine and iso-guanine.